Alpha-amylase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to alpha-amylase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/EP2016/060267 filed May 9, 2016, which claims priority or thebenefit under 35 U.S.C. 119 of Indian application No. 2336/CHE/2015filed May 8, 2015, the contents of which are fully incorporated hereinby reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to alpha-amylase variants, polynucleotidesencoding the variants, methods of producing the variants, and methods ofusing the variants.

Description of the Related Art

Alpha-amylases (α-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.) are agroup of enzymes that hydrolyzes starch, glycogen, and other relatedpolysaccharides by cleaving the internal α-1,4-glucosidic bonds. It hasbeen used for many years been in, e.g., laundry where is it well-knownthat alpha-amylases have a beneficial effect in removal of starchcontaining, or starch-based, stains. However, in other commercialapplications the enzyme has become important, such as in the initialstages (liquefaction) of starch processing, in textile desizing, inalcohol production and as cleaning agents in detergent compositions.

In recent years there has been a desire to improve the properties ofvarious amylases. In particular, the object of reducing the temperatureof the laundry in order to reduce the energy consumption has been ofprimary focus when referring to the householdcare sector. Thus, manyefforts have been put into finding improved alpha-amylase variants.

Regardless, there remains a need for alpha-amylase variants that possesaltered properties, and offer improved performance in various industrialapplications. Thus, the present invention provides such further improvedalpha-amylse variants with improved properties compared to its parent.

SUMMARY OF THE INVENTION

The present invention relates to an alpha-amylase variant of a parentpolypeptide having alpha-amylase activity, wherein the variant is amature form of an alpha-amylase having amylase activity, and comprisinga substitution at one or more positions selected from the groupconsisting of: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14,W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29,N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43,I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59,A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76,T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92,K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111,D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123,P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135,Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147,G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161,F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173,A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187,D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199,Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213,I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226,T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245,M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257,G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271,L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283,W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297,Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310,N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323,A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342,V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357,T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372,G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386,D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400,H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415,E416, N418, A419, S420, H421, P422, S424, G425, L426, A427, T428, I429,M430, S431, D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444,Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457,S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476,G477, V481, and W482; wherein the positions correspond to amino acidpositions in the amino acid sequence set forth in SEQ ID NO: 3; andwherein the substitution at the one or more positions provides analpha-amylase variant having an improvement factor of ≥1.0 for a measureof stability, and/or an improvement factor of ≥1.0 for a measure ofspecific activity.

The present invention also relates to polynucleotides encoding thevariants; nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides; and methods of producing the variants.

The present invention also relates to methods of improving washperformance and/or stability of a parent alpha-amylase.

DRAWINGS

FIG. 1 shows the Improvement Factor (IF) for a measure of specificactivity of the variants according to the present invention tested in aModel A detergent. The individual position modified is given as the Xaxis and the specific IF for each variant is given on the Y axis.

FIG. 2 shows the Improvement Factor (IF) for a measure of specificactivity of variants according to the present invention tested in aModel X detergent. The individual position modified is given as the Xaxis and the specific IF for each variant is given on the Y axis.

FIG. 3 shows the Improvement Factor (IF) for a measure of stability ofvariants according to the present invention tested in a Model Adetergent. The individual position modified is given as the X axis andthe specific IF for each variant is given on the Y axis.

CONVENTIONS FOR DESIGNATION OF VARIANTS

For purposes of the present invention, the mature polypeptide set forthin SEQ ID NO: 3 is used to determine the corresponding amino acidresidue in another alpha-amylase. The amino acid sequence of anotheralpha-amylase is aligned with the mature polypeptide set forth in SEQ IDNO: 3, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptide setforth in SEQ ID NO: 3 is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotheralpha-amylase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537:_39-64; Katoh and Toh, 2010,Bioinformatics 26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide set forthin SEQ ID NO: 3 such that traditional sequence-based comparison fails todetect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol.295: 613-615), other pairwise sequence comparison algorithms can beused. Greater sensitivity in sequence-based searching can be attainedusing search programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions: For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions: For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple Modifications: Variants comprising multiple modifications areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different Modifications: Where different modifications may be introducedat a position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants: “Tyr167Gly+Arg170Gly”,“Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a set of sequences. For ease, these arelisted below;

SEQ ID NO: 1 is the nucleotide sequence of a mature alpha-amylase(AAI10)

SEQ ID NO: 2 is the amino acid sequence of AAI10 including the signalpeptide, i.e. full-length.

SEQ ID NO: 3 is the amino acid sequence of the mature AAI10

SEQ ID NO: 4 is the amino acid sequence of the mature AAI10 comprising adouble deletion of the positions corresponding to positions G182 andD183 of SEQ ID NO: 3

SEQ ID NO: 5 is a PnMi4490 forward primer.

SEQ ID NO: 6 is a PnMi4491 forward primer.

Variants of the Invention

In one aspect, the present invention relates to variants of a parentpolypeptide having alpha-amylase activity. Thus, in particular aspect,the present invention relates to an alpha-amylase variant of a parentalpha-amylase having alpha-amylase activity, wherein said variant is amature form of an alpha-amylase having amylase activity, and comprisinga substitution at one or more positions selected from the groupconsisting of: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14,W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29,N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43,I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59,A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76,T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92,K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111,D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123,P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135,Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147,G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161,F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173,A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187,D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199,Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213,I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226,T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245,M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257,G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271,L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283,W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297,Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310,N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323,A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342,V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357,T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372,G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386,D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400,H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415,E416, N418, A419, S420, H421, P422, S424, G425, L426, A427, T428, I429,M430, S431, D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444,Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457,S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476,G477, V481, and W482; wherein the positions correspond to amino acidpositions in the amino acid sequence set forth in SEQ ID NO: 3; andwherein the substitution at the one or more positions provides analpha-amylase variant having an improvement factor of ≥1.0 for a measureof stability, and/or an improvement factor of ≥1.0 for a measure ofspecific activity.

It has been shown that variants according to the present invention whichcomprise a single substitution have an improved specific activity and/orstability compared to the parent alpha-amylase.

The term “amylase” or “alpha-amylase” as used herein, refers to anenzyme capable of catalyzing the degradation of starch. Generally,alpha-amylases (E.C. 3.2.1.1, α-D-(1->4)-glucan glucanohydrolase) areendo-acting enzymes that cleave the α-D(1->4) O-glycosidic linkageswithin the starch molecule in a random order.

The term “starch” as used herein, refers to any material comprised ofthe complex polysaccharide carbohydrates of plants, comprised of amyloseand amylopectin with the formula (C₆H₁₀O₅)_(x), wherein X can be anynumber. In particular, the term refers to plant-based materials, such asrice, barley, wheat, corn, rye, potato, and the like.

The term “alpha-amylase variant” as used herein, refers to a polypeptidehaving alpha-amylase activity comprising a modification, i.e., asubstitution, insertion, and/or deletion, at one or more (e.g., several)positions. A substitution means replacement of the amino acid occupyinga position with a different amino acid; a deletion means removal of theamino acid occupying a position; and an insertion means adding an aminoacid adjacent to and immediately following the amino acid occupying aposition, all as defined above. The variants of the present inventionhave at least 20%, e.g., at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 100% ofthe alpha-amylase activity of the amino acid sequence as set forth inSEQ ID NOs: 3. The alpha-amylase variant of the present inventioncomprises an amino acid sequence having at least 60%, such as at least65%, such as at least 70%, such as at least 75%, such as at least 80%,such as at least 85%, such as at least 90%, such as at least 95%, suchas at least 97%, such as at least 99%, but less than 100% sequenceidentity to any one of SEQ ID NOs: 2, 3, or 4.

The term “alpha-amylase activity” or “amylase activity” as used herein,refers to the activity of alpha-1,4-glucan-4-glucanohydrolases, E.C.3.2.1.1, which constitute a group of enzymes, catalyzing hydrolysis ofstarch and other linear and branched 1,4-glucosidic oligo- andpolysaccharides. Thus, the term “alpha-amylase” as used herein, refersto an enzyme that has alpha-amylase activity (Enzyme Class; EC 3.2.1.1)that hydrolyses alpha bonds of large, alpha-linked polysaccharides, suchas starch and glycogen, yielding glucose and maltose. The terms“alpha-amylase” and “amylase” may be used interchangeably and constitutethe same meaning and purpose within the scope of the present invention.For purposes of the present invention, alpha-amylase activity isdetermined according to the procedure described in the Examples. In oneembodiment, the variants of the present invention have at least 20%,e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 100% of the alpha-amylaseactivity of the polypeptide of SEQ ID NOs: 3 or 4; or the maturepolypeptide of SEQ ID NO: 2.

The term “parent alpha-amylase” as used herein, refers to analpha-amylase to which an alteration is made to produce enzyme variants.The alpha-amylase having an amino acid sequence as set forth in SEQ IDNOs: 2, 3 or 4, may e.g. be a parent for the variants of the presentinvention. Any polypeptide having at least 65%, such as at least 70%,such as at least 75%, such as at least 80%, such as at least 85%, suchat least 90%, such as at least 95%, such as at least 97%, such as atleast 99%, sequence identity to any one of SEQ ID NOs: 2, 3, or 4 mayalso be a parent polypeptide, such as a parent alpha-amylase, for thevariants of the present invention.

The parent alpha-amylase may be a fusion polypeptide or cleavable fusionpolypeptide. Such fusion polypeptide may consist of a subsequence of oneparent polypeptide and a subsequence of second parent polypeptide. Inparticular, a fusion polypeptide may consist of an A and B domain of onespecies of alpha-amylase and a C domain of another species ofalpha-amylase, and thereby providing a parent polypeptide to generate avariant of the present invention. Techniques for producing fusionpolypeptides are known in the art, and include ligating the codingsequences encoding the polypeptides so that they are in frame and thatexpression of the fusion polypeptide is under control of the samepromoter(s) and terminator. Fusion polypeptides may also be constructedusing intein technology in which fusion polypeptides are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

The terms “A domain”, “B domain” and “C domain” as used herein, refersto three distinct domains A, B and C, all part of the alpha-amylasestructure, see, e.g., Machius et al., 1995, J. Mol. Biol. 246: 545-559.Thus, an alpha-amylase, such as a parent polypeptide and a variantaccording to the invention, may comprise both an A, B, and C domain. Theterm “domain” means a region of a polypeptide that in itself forms adistinct and independent substructure of the whole molecule.Alpha-amylases consist of a beta/alpha-8 barrel harboring the activesite residues, which is denoted the A domain, a rather long loop betweenthe beta-sheet 3 and alpha-helix 3, which is denoted the B domain(together; “A and B domain”), and a C-domain and in some cases also acarbohydrate binding domain (e.g., WO 2005/001064; Machius et al.,supra).

The domains of an alpha-amylase may be determined by structure analysissuch as using crystallographically techniques. An alternative method fordetermining the domains of an alpha-amylase is by sequence alignment ofthe amino acid sequence of the alpha-amylase with another alpha-amylasefor which the domains have been determined. The sequence that alignswith, e.g., the C-domain sequence in the alpha-amylase for which theC-domain has been determined can be considered the C domain for thegiven alpha-amylase.

The term “A and B domain” as used herein, refers to two domains of analpha-amylase taken as one unit, whereas the C domain is another unit ofthe alpha-amylases. Thus, the amino acid sequence of the “A and Bdomain” is understood as one sequence or one part of a sequence of analpha-amylase comprising an “A and B domain” and other domains (such asthe C domain). In one embodiment, the A and B domain of a parentalpha-amylase correspond to amino acids 1 to 397 of the amino acidsequence as set forth in SEQ ID NO: 3.

The term “C domain” as used herein, refers to a domain of analpha-amylase as one unit. The “C domain” of an alpha-amylasecorresponds to amino acids 400 to 485 of SEQ ID NO: 3.

A fusion polypeptide may further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent polypeptide may be obtained from microorganisms of any genus.For purposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding a parentpolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a parent polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

In one embodiment, the parent alpha-amylase comprises or consists of theamino acid sequence set forth in SEQ ID NOs: 3, or 4. In anotherembodiment, the parent alpha-amylase comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In particular, the parent alpha-amylase maycomprise or consist of amino acids 1 to 485 of SEQ ID NO: 2.

In another embodiment, the parent alpha-amylase is a fragment of theamino acid sequence as set forth in SEQ ID NO: 3 containing at least350, such as at least 390 amino acid residues, e.g., at least 395 and atleast 397 amino acid residues of SEQ ID NO: 3.

In another embodiment, the parent alpha-amylase is an allelic variant ofthe mature polypeptide of SEQ ID NO: 3.

The term “sequence identity” as used herein, refers to the relatednessbetween two amino acid sequences or between two nucleotide sequences.For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the—nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

The term “fusion polypeptide” as used herein, refers to a polypeptidewhich comprises amino acid sequences originating from more than one,such as two, three, or four, species. Such a fusion polypeptide may havebeen generated by molecular techniques well-known to the skilled person.A fusion polypeptide of the present invention has alpha-amylaseactivity. In particular, a fusion polypeptide of the present invention,comprises e.g. an A and B domain of one alpha-amylase and a C domainfrom another alpha-amylase.

The term “mature polypeptide” and “mature amino acid sequence” as usedherein, refers to a polypeptide in its final form following translationand any post-translational modifications, such as N-terminal processing,C-terminal truncation, glycosylation, phosphorylation, etc. Alsocontemplated within the term “mature polypeptide” is that thesignalpeptide of the polypeptide has been cleaved off e.g. during anaturel maturation process within the cell expressing the polypeptide.In one aspect, the mature polypeptide is the amino acid sequence of SEQID NO:3. It is known in the art that a host cell may produce a mixtureof two of more different mature polypeptides (i.e., with a differentC-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide. It is also known in the art that different host cellsprocess polypeptides differently, and thus, one host cell expressing apolynucleotide may produce a different mature polypeptide (e.g., havinga different C-terminal and/or N-terminal amino acid) as compared toanother host cell expressing the same polynucleotide. In one embodiment,a mature polypeptide comprise up to 485 amino acid residues (e.g., aminoacids 1 to 485 of SEQ ID NO: 2). Thus, in one embodiment, the maturepolypeptide comprise up to 486 amino acid residues corresponding to theamino acid sequence set forth in SEQ ID NO:3.

The term “Improvement Factor” as used herein, refers to a quantitativeway of calculating the improvement of a particular property of a variantaccording to the present invention. Determination of the ImprovementFactor may be according to the following formula:

$\frac{{{Intensity}\mspace{14mu}{value}\mspace{14mu}{of}\mspace{14mu}{variant}} - {{Intensity}\mspace{14mu}{value}\mspace{14mu}{of}\mspace{14mu}{blank}}}{{{Intensity}\mspace{14mu}{value}\mspace{14mu}{of}\mspace{14mu}{parent}} - {{Intensity}\mspace{14mu}{value}\mspace{14mu}{of}\mspace{14mu}{blank}}}$

Other formulas may be used to determine the Improvement Factor. Theskilled person knows the presently presented formula as well asalternative ways of calculating the Improvement Factor.

According to the present invention, a value of 1.0 corresponds to thespecific activity observed for the parent alpha-amylase. A value above1.0 indicates an improvement of specific activity of the variant testedcompared to the parent alpha-amylase. Accordingly, any value of ≥1.0 isindicative for improvement of property, such as specific activity, ofthe variant compared to the parent alpha-amylase.

Similarly, the term Improvement Factor may be used when measuringstability, such as thermostability, in a detergent composition of avariant. Thus, the term Improvement Factor may also be used as anindicator for stability of a variant. According to the presentinvention, a value of 1.0 correspond to the stability observed for theparent alpha-amylase. A value above 1.0 indicates an improvement ofstability of the variant tested compared to the parent alpha-amylase.Accordingly, any value of ≥1.0 is indicative for improvement instability of the variant compared to the parent alpha-amylase.

The term “measure of stability” as used herein, refers to a measure ofenzymatic stability. Such measures of stability include stability indetergents, chelator stability, pH stability, towards protease,chloride, and/or bleach, and thermostability.

The term “stability” as used herein, refers to a the residual activityof a given variant which may be determined by incubating the variant ina model detergent composition preferably comprising chelating agentssuch as EDTA, EGTA, DTPA, DTMPA, MGDA or HEDP. Thus, the residualactivity is in particular relating to “chelator stability”, i.e. thevariant has been shown to have a given residual activity post incubationin a model detergent comprising a chelator. For example, as shown in theExample section, a variant may be tested in the a Model A detergentcomposition comprising EDTA in a final concentration of 0.1%. Theresidual activity may then be determined after a given period ofincubation time at a given temperature. E.g. by using EnzCheck orPhadebas assays described in the Example section, the residual activitycan be determined. Activity of the tested variant after the given timeand temperature may be compared to the activity of reference incubatedat low temperature, such as 4° C. and 0° C., for the same time period inthe same detergent composition. The lesser the difference between bothtreatments, the higher is the detergent stability. Similar tests may bedone using other detergents containing chelators, such as DTMPA, sodiumcitrate and HEDP. Similarly, surfactant stability may be determined in asimilar manner. Thus, the variant of the present invention maypreferably have an improved stability towards surfactants, such as anionsurfactants and LAS, wherein the surfactant stability may be determinedby the same assay as described above.

The term “improved storage stability” as used herein, refers to thestability of a given variant compared to the parent polypeptide, whereinresidual activity is measured upon storage for a given time andtemperature. Thus, the stability of a variant under storage conditionsis improved when compared to a reference, such as a parent polypeptide.

In one embodiment, the variant has an improved thermostability.

The term “improved thermostability” as used herein, refers to theimproved stability of a given variant compared to the parent polypeptidewherein the stability has been determined after incubation at a raisedtemperature for a given time period. E.g. a raised temperature may besuch as 50° C. or more.

In one embodiment, the improved stability is determined by a methodcomprising the steps of;

-   -   a) incubating an alpha-amylase variant sample and a parent        alpha-amylase sample, respectively, in a model detergent        composition, such as Model A, Model J, Model T, or Model X, for        a period of time;    -   b) measuring the activity of the variant alpha-amylase and the        parent polypeptide, respectively; and    -   c) calculating the residual activity of the samples.

If the stability is determined in a model powder detergent, such asModel T or Model X, the stability may preferably be measured byincubation in a wash solution of said model powder detergent, e.g. thepowder is dissolved in water wherein said variant is incubated.

In a further embodiment, the improved stability is determined by amethod comprising the steps of;

-   -   a) incubating an alpha-amylase variant sample and a parent        polypeptide sample, respectively, in a model detergent        composition, such as Model A, Model J, Model T, or Model X, at        40° C. to 60° C. for 2 to 168 hrs;    -   b) measuring the activity of the variant alpha-amylase and the        parent polypeptide, respectively; and    -   c) calculating the residual activity of the samples as the        average of activity in the samples relative to the average of        the activity to frozen control samples.

The term “measure of specific activity” as used herein, refers to ameasure of enzymatic acitivity. Such measures of activity include washperformance at pH 8.0 at 25° C. and activity using a syntheticsubstrate, such as Phadebas tables (Magle Life Sciences).

The term “specific activity” as used herein, refers to the number ofmoles of substrate converted to product by an enzyme preparation perunit time under specific conditions. Specific activity is expressed asunits (U)/mg of protein. Specific activity may be determined asdescribed in the Example 2.

The term “wash performance” as used herein, refers to an enzyme'sability to remove starch or starch-containing stains present on theobject to be cleaned during e.g. laundry or hard surface cleaning, suchas dish wash. The term “wash performance” includes cleaning in generale.g. hard surface cleaning as in dish wash, but also wash performance ontextiles such as laundry, and also industrial and institutionalcleaning. The wash performance may be quantified by calculating theso-called Intensity value, and results may be displayed as “ImprovementFactor” (IF). Wash performance may be determined as in described in theExamples herein.

The term “Intensity value” as used herein, refers to the washperformance measured as the brightness expressed as the intensity of thelight reflected from the sample when illuminated with white light. Whenthe sample is stained the intensity of the reflected light is lower,than that of a clean sample. Therefore the intensity of the reflectedlight can be used to measure wash performance, where a higher intensityvalue correlates with higher wash performance.

Color measurements are made with a professional flatbed scanner (KodakiQsmart, Kodak) used to capture an image of the washed textile.

To extract a value for the light intensity from the scanned images,24-bit pixel values from the image are converted into values for red,green and blue (RGB). The intensity value (Int) is calculated by addingthe RGB values together as vectors and then taking the length of theresulting vector:Int=√{square root over (r ² +g ² +b ²)}

The term “improved wash performance” as used herein, refers to animprovement of the wash performance of an alpha-amylase of the presentinvention relative to the wash performance of the parent polypeptide.Improved wash performance may be measured by comparing of the so-calledIntensity value and calculating the Improvement Factor (IF). Theimproved wash performance is determined according to the section “Washperformance of alpha-amylases using Automatic Mechanical Stress Assay”and using model detergent J at 15° C. Other model detergents may beused, such as Model detergent A, Model detergent X or Model detergent T.

Thus, in one embodiment, the improved wash performance is determined bya method comprising the steps of;

-   -   a) washing a fabric stained with starch with an alpha-amylase        variant and a parent polypeptide sample added, respectively, to        a model detergent composition, such as Model A, Model J, Model        T, or Model X;    -   b) measuring the intensity of light reflected from the sample        when illuminated with white light; and    -   c) optionally, calculating the improvement factor (IF) as the        ration of delta intensity of the alpha-amylase sample over the        delta intensity of the parent polypeptide sample.

In one embodiment, the improved wash performance is determined by amethod comprising the steps of;

-   -   a) washing a fabric stained with starch with an alpha-amylase        variant and a parent polypeptide sample added, respectively, to        a model detergent composition, such as Model A, Model J, Model        T, or Model X, for 20 minutes at 15° C. and 30° C.;    -   b) measuring the intensity of light reflected from the sample        when illuminated with white light; and    -   c) optionally, calculating the improvement factor (IF) as the        ration of delta intensity of the alpha-amylase sample over the        delta intensity of the parent polypeptide sample.

As can be seen from the data obtained in Example 4 and FIG. 1-3, a meanhit value for each tested position show that many variants having amodification in one of the positions have an IF of at least 1.0 for atleast one of the tested conditions, i.e. specific activity in Model A orModel X, or stability.

The term “mean hit value” as used herein, refers to a mean value of theImprovement Factor measured for all the generated specific variantshaving an IF of more than 1.0 for each of the tested conditions, i.e.specific activity tested in Model A or Model X detergent compositions orthe stability of the variants. Thus, the Improvement Factors for eachindividual variant in a particular position have been added up anddivided by the number of variants for the particular position, andthereby providing the mean hit value.

In one embodiment, the variant comprises one or more of thesubstitutions selected from the group consisting of; H1A, H1G, H1V, H1C,H1S, H1L, H2T, H2G, H2L, H2M, H2E, H2P, D3A, D3C, D3R, D3Y, D3W, D3S,D3F, G4D, G4Q, G4R, T5L, T5Q, T5E, T5V, T5G, T5F, T5W, G7F, G7E, G7V,G7D, G7R, G7A, G7L, G7S, G7N, T8A, T8S, I9C, I9M, I9L, M10L, Q11C, Q11L,Q11V, Q11M, Y12V, Y12A, Y12F, F13I, F13Y, E14M, E14L, E14S, E14Q, E14FW15G, W15F, W15S, W15I, W15H, N16G, N16Q, N16R, V17Q, V17A, V17T, V17L,V17D, V17G, V17E, P18L, N19G, N19S, N19Y, N19T, N19V, D20R, Q22C, Q22A,Q22N, Q22S, Q22M, Q22T, Q22E, Q22Y, H23W, W24H, W24F, N25V, N25G, N25E,N25E, N25S, N25R, N25L, N25T, R26L, R26G, R26D, R26I, R26S, R26W, R26A,R26F, R26M, R26E, L27A, L27M, H28E, H28A, H28W, H28K, H28I, H28R, H28M,N29G, N29C, N29L, N29K, N29V, N29W, N29A, N30S, N30H, N30L, N30T, N30E,N30R, A31S, A31W, A31V, A31G, A31R, Q32G, Q32L, Q32A, Q32D, N33F, N33A,N33R, N33D, L34Y, L34F, K35L, K35E, K35W, K35Q, N36R, N36V, N36Q, N36A,N36P, N36W, N36E, N36T, A37R, A37T, A37S, A37G, A37E, G38N, G38M, G38Y,G38K, 139L, I39M, I39V, T40D, T40A, T40Q, T40E, T40S, A41Q, A41E, A41N,A41M, A41D, I42A, I42V, I42L, W43R, I44T, I44L, I44V, P45A, P46G, A47S,A47G, A47T, W48C, W48I, W48L, W48G, W48M, W48P, W48Q, W48H, W48A, W48Y,W48F, K49Q, G50S, G50N, G50P, T51S, T51G, T51C, T51P, T51L, T51A, S52V,S52M, S52I, S52L, S52A, S52D, S52K, S52N, S52E, S52T, S52C, S52F, Q53L,Q53V, Q53G, Q53M, N54G, N54K, N54V, N54T, N54L, N54I, N54S, D55G, D55V,D55I, V56L, V56M, V56T, G59E, G59A, G59S, A60S, A60W, A60L, A60G, A60V,A60F, A60T, Y61F, L63P, L63I, L63V, L63H, L63K, L63C, L63G, L63A, Y64W,Y64S, Y64V, Y64I, Y64H, Y64M, Y64C, Y64R, Y64G, Y64L, Y64A, L66M, E68D,E68S, E68M, E68C, E68T, E68V, E68Q, E68Y, F69H, F69W, N70G, N70A, N70T,N70S, N70E, Q71E, Q71N, Q71D, Q71I, Q71V, K72Y, K72R, G73V, G73L, G73E,G73C, T74L, T74V, T74N, V75T, V75G, V75E, R76S, R76G, R76A, R76V, T77A,T77S, T77V, K78L, K78T, K78A, T81W, T81L, T81G, K82A, K82G, A83G, A83E,A83Q, A83R, A83S, A83F, A83V, E84F, E84D, E84M, E84W, L85V, E86L, E86D,E86W, E86R, R87M, R87A, R87E, A88S, A88G, A88V, I89A, I89P, I89H, I89L,R90L, R90G, S91A, S91G, S91E, S91V, S91T, L92C, L92F, K93W, K93G, K93L,N95G, N95E, N95L, N95S, N95W, N95C, N95D, N95R, G96Q, G96R, I97M, I97V,I97L, Q98V, Q98L, Q98C, Q98M, Q98T, Q98F, Q98W, Q98P, Q98H, V99I, V99L,V99S, V99T, V99G, V99E, Y100W, Y100V, Y100F, V103G, V103A, V103S, V103T,H107Q, H107M, K108V, G109A, G109S, G109F, G109D, G110C, A111S, D112E,D112K, D112V, D112F, D112C, F113M, F113V, F113E, F113L, F113C, F113D,F113K, F113Q, F133Y, T114S, T114V, T114L, E115C, E115A, R116Q, R116N,R116L, R116I, R116D, R116T, R116G, R116E, R116S, R116C, R116V, R116F,R116K, R116H, R116W, V117G, V117C, V117S, V117W, V117A, V117E, V117L,V117I, V117M, Q118E, I118F, Q118R, Q118V, Q118G, A119Y, A119W, V120I,V120D, V120L, V120C, V120A, V120N, E121V, E121M, E121D, E121P, E121G,V122E, V122P, V122L, V122I, V122F, N123G, N123A, N123E, N123W, N123T,N123P, N123K, N123V, P124R, P124G, Q125E, Q125T, N126G, N126A, N126E,N126T, N126R, R127G, R127S, R127C, R127W, R127L, R127V, R127F, R127Y,N128S, N128E, N128C, N128A, N128P, N128G, Q129L, Q129E, Q129C, E130C,V131L, V131E, V131I, S132A, S132H, S132P, S132C, S132E, S132D, S132V,S132Q, S132N, G133T, G133L, G133A, G133D, G133S, G133V, G133I, G133E,G133R, T134V, T134D, T134E, Y135L, Y135V, Y135S, Y135R, Y135T, Y135W,Y135K, Y135G, Y135P, Y135M, Y135D, Y135H, Y135F, Y135E, Q136E, Q136W,Q136G, I137A, I137S, I137G, I137V, E138A, E138L, E138Y, E138T, A139S,A139M, A139G, A139V, A139T, A139P, W140H, W140Y, W140S, W140A, T141C,T141G, T141S, T141M, G142D, G142E, G142A, G142R, F143M, F143L, F143I,N144C, N144G, N144R, N144M, N144K, F145A, F145C, F145S, F145N, F145Q,F145T, F145H, P146L, P146S, P146V, P146C, P146M, P146T, G147V, G147P,G147D, G147S, G147I, G147L, G147C, G149C, G149Y, G149L, G149A, N150C,N150D, N150E, Q151L, Q151T, Q151C, Q151I, S153C, S153G, S153T, S154M,S154E, S154A, S154V, F155T, F155H, F155L, F155W, K156Q, K156F, K156L,K156S, K156G, K156Y, K156V, K156A, K156D, K156W, K156P, K156E, K156C,K156N, K156T, W157L, R158S, R158M, R158A, R158G, R158Q, R158W, R158L,R158T, R158V, R158E, R158Y, W159N, W159G, W159T, W159C, W159E, W159D,W159S, W159H, Y160M, Y160A, Y160Q, Y160T, Y160S, Y160V, Y160G, Y160I,Y160L, Y160E, Y160W, Y160R, Y160H, H161N, H161C, F162M, F162L, F162Y,F162I, D163A, D163R, D163S, D163K, D163L, G164S, G164T, G164C, T165L,T165S, T165I, T165M, T165G, T165V, T165E, D166S, D166T, D166N, W167G,W167Y, W167D, W167F, D168M, D168S, D168G, Q169L, Q169A, Q169S, Q169V,Q169F, Q169E, S170A, S170V, S170Q, S170W, S170G, S170F, S170T, S170L,R171A, R171F, R171S, R171K, R171V, R171M, Q172E, Q172L, Q172S, Q172R,Q172G, Q172W, Q172C, L172K, L173I, L173G, I173N, L173M, L173S, L173Y,L173V, L173A, L174F, A174V, A174G, A174K, A174L, A174M, A174S, N175G,N175L, N175V, N175I, N175S, R176W, R176E, R176G, R176M, R176M, R176T,R176K, R176Y, I177L, I177S, I177G, Y178V, Y178R, Y178K, Y178W, K179L,K179C, K179E, K179S, K179W, K179A, K179G, K179T, K179F, F180A, F180L,R181A, R181G, R181K, R181P, R181S, R181V, R181Q, R181E, R181D, G184L,G184R, G184V, G184T, G184D, K185G, K185A, K185D, K185N, K185V, A186S,A186D, A186C, A186T, A186Q, A186V, A186N, A186E, A186H, W187G, D188R,D188Q, D188G, D188M, D188L, D188V, W189R, W189A, W189G, W189C, E190D,E190N, E190A, V191M, V191A, D192E, D192Q, D192S, T193K, T193V, T193C,T193P, T193S, T193G, T193D, T193R, E194S, E194D, E194L, E194Q, N195R,N195A, N195F, N195T, G196A, G196V, G196P, N197G, N197V, N197Y, N197C,N197D, Y198W, Y198A, Y198G, D199G, D199S, D199A, Y200W, Y200F, Y200C,Y200V, Y200T, Y200L, Y200A, Y200G, Y200S, M202T, M202L, Y203L, Y203W,Y203F, Y203G, Y203C, Y203I, Y203Q, Y203H, Y203T, Y203M, Y203V, Y203S,A204P, A204C, A204R, A204H, A204W, A204F, A204L, A204Y, A204D, A204G,D205C, D205A, D205L, D205R, V206H, V206Q, V206P, V206A, V206S, V206K,V206L, V206I, V206M, V206F, V206Y, M208R, M208P, M208L, M208C, D209C,D209E, D209H, D209F, D209Y, D209S, D209M, D209V, D209L, D209I, D209Q,H210V, H210F, H210A, H210W, H210L, H210G, H210D, H210E, H210Q, H210N,P211G, P211W, P211C, E212G, E212I, E212F, E212M, E212D, E212K, E212Y,E212R, E212C, V213M, V213T, V213G, V213C, V213W, V213R, V213F, I214P,I214V, N215C, N215D, N215V, N215T, L217V, L217S, L217T, L217G, L217C,L217P, L217A, L217W, N218S, N218D, N218R, N218L, N218M, R219L, R219W,R219S, R219A, R219H, R219I, R219M, R219T, R219E, R219Y, R219D, R219C,W220I, W220P, W220E, W220R, W220A, W220S, W220L, G221S, G221A, V222F,V222C, V222E, V222L, W223Y, W223F, W223L, Y224L, A225K, N226C, N226Q,N226M, N226E, N226K, N226R, T227S, T227M, T227C, T227L, T227Y, T227R,L228A, L228F, L228M, N229T, N229S, L230S, L230A, L230T, L230V, D231E,D231C, D231G, D231P, F233A, F233L, F233T, F233P, F233M, V238C, V238W,V238A, H240W, I241L, K242A, K242C, K242Y, K242P, K242W, K242G, K242L,K242R, K242S, K242E, K242Q, K242H, S244Q, F245G, F245C, F245V, F245D,F245A, M246L, M246F, M246V, M246I, R247Y, R247G, R247M, D248G, D248R,W249L, W249F, L250W, L250T, L250A, L250C, L250Y, L250F, G251K, G251M,G251Q, H252E, H252W, H252C, H252V, H252T, H252F, V253S, V253W, V253A,V253L, R254Q, R254S, G255C, G255E, G255L, Q256P, T257V, G258V, G258C,G258I, G258P, G258L, G258D, G258F, K259V, K259Q, K259I, K259L, K259A,K259S, K259D, K259C, N260E, N260C, N260W, N260D, N260S, N260R, N260G,L261G, L261D, L261T, F262Q, F262L, F262E, F262R, F262P, F262V, A263L,A263C, V264T, V264L, Y267V, Y267L, W268V, W268Y, K269W, K269L, K269Q,K269R, N270D, N270A, N270M, N270G, D271E, D271V, D271C, L272M, G273W,G273R, G273V, G273E, A274S, A274D, A274E, L275G, L275E, L275A, L275R,L275K, E276R, E276L, N277D, Y278G, Y278S, L279V, L279Q, L279G, L279M,S280C, S280R, S280E, S280D, K281L, K281A, K281W, K281G, T282G, T282A,T282C, T282R, T282I, N283G, N283E, N283Q, N283V, N283A, W284T, W284I,W284S, W284E, W284C, W284V, W284A, T285E, T285F, T285S, T285P, T285W,T285M, M286D, M286L, M286C, S287T, S287E, S287V, S287Q, S287P, S287L,S287W, S287C, A288L, A288Y, A288V, V291T, P292A, P292Q, P292L, P292R,P292G, L293A, L293E, L293T, L293G, L293Q, L293Y, H294G, H294A, H294W,H294Q, H294S, Y295H, Y295S, Y295I, Y295A, N296A, N296Y, N296M, N296F,N296H, N296S, N296K, L297S, L297W, L297H, Y298V, Y298P, Q299S, Q299L,Q299R, Q299I, Q299P, Q299A, Q299H, A300V, A300C, S301A, S301R, S301Q,N302I, N302T, N302M, N302S, S303I, S303E, S303A, S303M, S304I, S304P,S304G, S304L, S304F, S304A, S304M, G305S, G305V, G305L, G305F, G305I,G305Q, N306V, N306L, N306D, N306S, N306T, Y307F, Y307W, M309L, R310A,R310T, N311C, N311Y, N311L, N311R, L312F, L312V, L312I, L313S, L313M,L313I, N314Y, N314D, N314F, G315V, G315I, G315T, G315R, G315F, T316K,T316S, T316C, T316G, V318C, V318E, V318L, V318A, Q319L, Q319I, Q319A,Q319H, Q319D, Q319F, Q319P, R320C, R320V, R320L, R320A, R320S, H321T,H321E, H321V, H321C, H321S, H321G, H321R, P322E, P322C, S323N, A325V,A325S, V326I, V326G, V326A, V326C, V326T, T327S, T327L, T327V, T327E,T327Q, F328M, F328C, V329A, 329S, V329C, V329I, N331C, N331V, N331I,T334S, T334A, P336M, P336E, P336D, P336R, P336L, P336S, P336K, P336W,G337T, G337V, G337C, G337M, G337F, E338A, E338H, E338T, E341C, E341D,E341I, E341M, E341F, E341K, E341S, E341P, E341T, S342G, S342T, S342A,V344S, V344L, Q345K, Q345V, Q345I, Q345L, Q345C, Q345W, Q345N, G346H,G346A, G346L, G346N, G346R, G346Q, G346V, G346S, W347L, W347R, W347V,W347C, F348Q, F348S, F348C, F348P, F348M, K349G, K349A, K349Y, K349L,K349S, P350S, P350Q, P350L, P350G, P350D, P350E, L351W, L351M, L351S,L351Q, L351H, A352S, A352C, A354C, T355V, T355A, T355L, T355Y, T355F,L357C, L357S, L357T, L357H, T358V, T358F, T358S, T358L, E360S, E360D,E360C, E360G, E360A, E360M, Q361P, Q361R, Q361V, Q361A, Q361E, Q361G,Q361D, Q361S, G362Y, G362R, G362C, G362N, G362M, G362V, Y363Q, Y363L,Y363I, Y363A, Y363V, Y363R, Q365D, Q365M, Q365L, Q365T, Q365C, Q365S,V366L, V366A, V366I, V366C, F367L, F367S, F367A, Y368M, Y368T, Y368H,Y368A, Y368S, Y368Q, Y368I, Y368L, Y368V, G369K, G369S, G369E, G369A,G369R, G369I, G369L, D370A, D370T, Y372A, Y372S, Y372I, Y372L, Y372G,G373C, G373S, G373R, G373T, G373Q, G373W, I374Y, I374L, I374S, I374M,P375L, P375A, P375T, P375V, P375R, P375E, P375S, S376A, S376N, S376P,S376W, S376R, S376L, S376E, S376T, S376V, S376I, S376Q, S376F, D377C,D377L, D377V, D377T, D377S, D377G, V379L, V379F, V379P, V379S, P380L,P380E, P380A, P380V, P380G, P380I, P380Y, S381N, S381L, S381A, S381I,S381V, Y382I, Y382A, Y382E, Y382L, Y382V, Y382R, Y382K, R383G, R383S,R383P, R383V, Q384A, Q384P, Q384L, Q384H, Q384R, Q384Y, Q384S, Q384V,Q384T, Q384G, Q384E, I386T, I386L, I386M, I386W, I386V, D387H, D387V,D387Q, D387L, P388G, P388T, P388M, P388V, P388W, P388K, P388L, P388S,P388R, L389S, L389H, L389A, L389V, L389F, L389M, L390S, L390F, L390I,L390G, L390V, L390T, L390N, L390H, L390C, K391L, K391Y, K391A, K391Q,A392I, A392M, A392T, Q394S, Q394R, Q394L, Q394T, Q394N, Q394M, Q395W,Q395N, Q395L, Q395E, Q395K, Q395R, Q395V, Q395S, Q395M, Y396T, Y396L,Y396R, Y396W, A397C, A397G, A397S, Y398K, Y398S, Y398C, Y398N, R400L,R400C, R400K, R400S, R400M, R400E, R400I, H402V, H402E, H402M, H402A,H402Y, H402S, H402P, H402L, H402D, H402R, H402T, D403E, D403P, D403Q,D403I, Y404F, Y404W, Y404K, F405C, F405V, F405I, F405P, F405W, F405G,F405A, D406E, D406V, D406T, D406M, D406S, H407C, H407D, W408R, W408G,W408D, W408P, W408C, W408Y, D409G, V410I, V410Q, V410E, V410L, I411A,I411L, I411V, T414A, T414V, R415F, R415L, R415A, R415Q, E416C, E416V,E416L, E416A, E416Q, N418L, N418A, N418C, N418M, N418G, N418R, N418F,A419E, A419C, A419M, A419Q, A419G, A419V, A419L, A419R, S420E, S420T,S420V, S420M, H421V, H421C, H421A, H421R, H421T, P422Y, P422H, P422D,P422R, S424C, S424A, S424L, G425A, G425V, L426S, L426A, A427V, A427S,T428L, T428N, T428A, T428G, I429V, 1429A, M430R, M430L, M430A, M430T,S431A, S431T, S431F, D432Q, D432P, G435E, G435N, G435M, G435I, G435S,G436L, G436M, G436E, G436T, S437G, S437Q, S437L, K438T, W439C, W439H,W439E, W439A, W439R, W439T, W439N, W439T, W439R, W439N, M440L, Y441N,Y441H, Y441Q, Y441K, V442I, 444A, R444S, R444L, R444P, R444V, Q445L,Q445S, Q445C, Q445E, Q445R, K446Q, K446G, K446T, K446P, A447G, A447Q,A447E, A447T, A447R, A447S, G448S, G448R, V450A, V450T, V450Q, V450R,W451L, H452A, H452T, H452C, H452G, H452Y, H452R, H452V, D453E, M454R,M454L, M454V, M454Q, M454S, T455L, T455S, G456V, G456R, G456Y, G456K,G456L, N457V, N457I, N457G, N457R, S459E, S459V, S459C, S459P, S459M,S459T, S459Q, S459R, G460S, G460D, G460E, T463C, T463V, T463L, T463D,T463H, T463M, T463A, T463Q, I464V, N465G, N465Q, N465L, N465R, Q466L,Q466C, Q466E, D467V, D467C, W469T, W469M, W469E, W469N, H471Q, F473Y,F473C, F473R, F473E, F473G, F473T, F473V, N475T, N475H, N475G, G476F,G476Y, G476K, G477Q, G477F, G477K, G477L, V481Q, V481S, and W482F,wherein the numbering is according to the amino acid sequence set forthin SEQ ID NO: 3, and wherein the substitution at the one or morepositions provides a variant having an IF of ≥1.0 for a measure ofstability and/or an IF of ≥1.0 for a measure of specific activity.

The term “corresponding to” as used herein, refers to way of determiningthe specific amino acid of a sequence wherein reference is made to aspecific amino acid sequence. E.g. for the purposes of the presentinvention, when references are made to specific amino acid positions,the skilled person would be able to align another amino acid sequence tosaid amino acid sequence that reference has been made to, in order todetermine which specific amino acid may be of interest in said anotheramino acid sequence. Alignment of another amino acid sequence with e.g.the sequence as set forth in SEQ ID NO: 3, or any other sequence listedherein, has been described elsewhere herein. Alternative alignmentmethods may be used, and are well-known for the skilled person.

In one embodiment, the parent alpha-amylase has at least 85%, such as atleast 90%, such as at least 95%, such as 97%, such as at least 98%,sequence identity with the amino acid sequence set forth in SEQ ID NO:3. In another embodiment, the parent alpha-amylase has at least 85%,such as at least 90%, such as at least 95%, such as 97%, such as atleast 98%, sequence identity with the amino acid sequence set forth inSEQ ID NO: 4.

In one embodiment, the parent alpha-amylase consists of or comprises theamino acid sequence set forth in SEQ ID NOs: 3 or 4.

The present invention provides variants of such parent alpha-amylases.

Accordingly, the variants of the present invention comprise an aminoacid sequence having at least 60%, such as at least 65%, such as atleast 70%, such as at least 75%, such as at least 80%, such as at least85%, such as at least 90%, such as at least 95%, such as at least 97%,such as at least 98%, such as at least 99%, but less than 100% sequenceidentity to the amino acid sequences set forth in SEQ ID NOs: 3 and 4.

The variants according to the present invention may comprise othermodifications than a substitution, such as an insertion or a deletion.Such modifications may also be termed “alterations”. Thus, the term“alteration” as used herein, refers to both substitutions and deletionsof amino acid within the amino acid sequence of a polypeptide. The terms“alteration” and “modification” may be used interchangeably herein. Thisshould not be understood as any limitation and thus, the termsconstitute the same meaning and purpose unless explicitly statedotherwise.

In one embodiment, the variant comprises a substitution at one or morepositions selected from the group consisting of; H1, H2, D3, G4, T5, G7,T8, I9, Q11, Y12, F13, E14, W15, N16, P18, N19, D20, Q22, W24, N25, R26,L27, H28, N29, N30, A31, Q32, L34, K35, N36, A37, 139, T40, A41, W43,I44, P45, P46, A47, W48, G50, T51, S52, Q53, N54, D55, V56, G59, A60,Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77,K78, K82, A83, E84, L85, R87, A88, I89, R90, S91, L92, K93, N95, G96,I97, Q98, V99, Y100, V103, H107, K108, G109, D112, F113, E115, R116,V117, A119, V120, E121, V122, N123, Q125, N126, R127, N128, Q129, E130,V131, S132, G133, T134, Y135, I137, A139, W140, T141, G142, F143, N144,F145, P146, G147, G149, N150, Q151, S153, F155, K156, W157, R158, W159,Y160, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172,L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186,W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198,D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212,V213, I214, N215, L217, R219, V222, W223, A225, N226, T227, L228, N229,L230, D231, F233, V238, I241, K242, F245, M246, W249, L250, H252, R254,G255, T257, G258, K259, N260, L261, F262, A263, Y267, W268, K269, N270,D271, G273, A274, Y278, L279, S280, K281, T282, N283, W284, T285, M286,S287, A288, V291, P292, L293, H294, N296, L297, Q299, A300, S301, S303,S304, G305, N306, L312, L313, N314, G315, T316, V318, Q319, R320, H321,P322, A325, P336, G337, E341, S342, V344, Q345, G346, W347, F348, K349,P350, L351, A352, L357, T358, Q361, G362, Y363, Q365, V366, Y368, G369,D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383,Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397,R400, H402, F405, D406, H407, W408, V410, T414, R415, E416, N418, A419,H421, G425, A427, T428, I429, M430, S431, G435, G436, S437, W439, M440,V442, R444, Q445, A447, G448, H452, M454, T455, G456, N457, S459, G460,T463, N465, D467, W469, H471, F473, N475, G477, and V481; wherein thepositions correspond to amino acid positions in the amino acid sequenceset forth in SEQ ID NO: 3, which one or more substitutions provides animprovement factor of ≥1.2 for a measure of specific activity in Model Adetergent composition, and optionally, an improvement factor of ≥1.0 fora measure of stability.

In a particular embodiment, the variant comprises one or more of thesubstitutions selected from the group consisting of; H1V, H1G, H1S, H1C,H2T, H2L, H2M, H2E, H2P, H2G, D3A, G4Q, G4D, G4R, T5Q, T5G, T5E, T5V,G7F, G7V, G7E, G7E, G7D, G7R, T8S, I9M, I9L, Q11L, Q11V, Y12V, Y12A,Y12F, F13Y, F13I, E14L, E14M, E14S, W15G, W15S, W15F, N16G, N16Q, P18L,N19G, N19S, N19Y, N19T, N19V, D20R, Q22C, Q22A, Q22N, Q22S, Q22M, Q22T,Q22E, W24F, N25V, N25S, N25G, N25E, N25E, R26L, R26G, R26D, R26M, R26I,R26S, R26W, R26A, R26F, L27A, L27M, H28A, H28E, H28I, N29G, N29C, N29L,N30S, N30H, N30L, N30T, N30E, A31S, A31V, Q32D, Q32G, Q32L, Q32A, L34F,L34Y, K35L, K35E, K35W, K35Q, N36R, N36Q, N36A, N36E, A37T, A37S, I39M,I39L, T40E, T40A, A41E, A41N, A41M, A41Q, A41D, W43R, I44T, P45A, P46G,W48G, W48L, W48C, W48I, W48M, W48P, W48Q, W48Y, W48F, G50P, G50S, G50N,T51S, T51G, T51P, T51L, T51C, S52M, S52V, S52I, S52L, Q53V, Q53L, Q53G,N54G, N54K, N54V, D55V, D55I, V56L, V56M, G59E, G59A, A60S, A60W, A60L,A60G, A60T, Y61F, L63V, L63P, L63I, L63H, L63K, Y64W, Y64S, Y64V, Y64I,Y64H, Y64M, Y64C, Y64R, Y64G, Y64L, Y64A, L66M, E68D, F69H, F69W, N70G,N70A, N70T, N70S, N70E, Q71N, Q71E, Q71D, Q71I, Q71V, K72Y, K72R, G73V,G73L, G73E, T74V, T74L, V75T, V75G, R76S, R76G, R76A, T77S, K78L, K78T,K78A, K82A, A83G, A83E, E84D, L85V, R87M, R87A, R87E, A88V, I89H, I89P,I89A, R90L, R90G, S91E, S91A, S91G, L92C, K93W, K93G, K93L, N95L, N95W,N95C, N95E, N95S, N95G, G96Q, I97V, Q98C, Q98M, Q98T, Q98V, Q98L, V99I,V99L, V99S, V99T, Y100V, Y100F, Y100W, V103G, H107Q, K108V, G109A,G109S, D112E, F113L, F113M, F113V, F113E, F113C, F113D, F113Y, E115A,R116N, R116L, R116I, R116D, R116T, R116G, R116E, R116S, R116C, R116V,R116Q, R116F, V117C, V117L, V117S, V117W, V117A, V117G, V117E, A119Y,V120D, V120L, E121P, E121G, E121V, E121M, E121D, V122E, V122P, V122I,N123G, N123A, N123E, N123W, N123T, N123P, Q125E, Q125T, N126E, N126G,N126A, R127G, R127S, R127C, R127W, R127L, R127V, R127F, R127Y, N128C,N128S, N128E, Q129L, Q129C, Q129E, E130C, V131L, V131E, S132A, S132H,S132P, S132C, S132E, S132D, S132N, G133S, G133T, G133L, G133V, G133I,G133E, G133A, G133D, T134V, T134D, T134E, Y135L, Y135V, Y135S, Y135R,Y135T, Y135W, Y135K, Y135G, Y135P, Y135M, Y135D, Y135H, Y135F, Y135E,I137A, I137S, I137G, A139M, A139S, W140Y, W140S, W140A, W140H, T141M,T141S, T141C, T141G, G142D, G142E, F143M, F143L, N144C, F145C, F145S,F145A, P146S, P146V, P146C, P146L, G147V, G147P, G147D, G147S, G147I,G147L, G147C, G149C, N150C, N150D, N150E, Q151T, Q151C, Q151L, S153C,F155H, F155T, F155L, K156Y, K156V, K156A, K156D, K156W, K156P, K156E,K156C, K156F, K156Q, K156L, K156S, K156G, W157L, R158M, R158A, R158G,R158Q, R158W, R158L, R158T, R158V, R158E, R158Y, R158S, W159N, W159G,W159T, W159C, W159E, W159D, W159S, W159H, Y160Q, Y160M, Y160T, Y160A,Y160S, Y160V, Y160G, Y160I, Y160L, Y160E, F162M, F162L, F162Y, F162I,D163K, D163A, D163R, D163L, G164S, G164T, G164C, T165L, T165M, T165S,T165G, T165I, D166T, D166N, D166S, W167Y, W167G, W167D, D168M, D168S,D168G, Q169A, Q169L, Q169E, S170A, S170V, R171F, R171S, R171K, R171V,R171A, Q172S, Q172L, Q172E, L173G, L173N, L173M, L173S, L173Y, L173V,L173I, A174G, A174V, N175L, N175V, N175I, N175G, R176E, R176W, R176G,R176M, I177L, I177S, I177G, Y178V, Y178R, Y178K, K179C, K179E, K179S,K179W, K179A, K179G, K179T, K179F, K179L, F180A, F180L, R181K, R181P,R181S, R181V, R181Q, R181E, R181A, R181G, G184L, G184R, G184V, G184D,K185A, K185D, K185N, K185V, K185G, A186C, A186S, A186D, W187G, D188Q,D188G, D188R, D188M, D188L, D188V, W189R, W189A, W189G, W189C, E190D,E190N, E190A, V191M, V191A, D192Q, D192S, T193K, T193V, T193C, T193P,T193S, E194D, E194L, E194Q, N195R, N195A, G196A, G196V, N197G, N197V,N197Y, N197C, N197D, Y198W, D199G, D199S, D199A, Y200W, Y200F, Y200C,Y200V, Y200T, Y200L, Y200A, Y200G, Y200S, M202T, Y203F, Y203G, Y203C,Y203I, Y203Q, Y203H, Y203T, Y203M, Y203S, Y203V, Y203W, Y203L, A204P,A204R, A204C, A204H, A204W, A204F, A204L, A204Y, A204D, A204G, D205C,V206H, V206Q, V206P, M208C, D209E, D209H, D209F, D209Y, D209S, D209M,D209V, H210V, H210A, H210W, H210L, H210G, H210D, H210E, H210N, P211G,P211W, P211C, E212F, E212M, E212D, E212G, E212I, V213W, I214P, N215D,N215V, N215C, L217C, L217P, L217T, L217G, L217S, R219H, R219W, R219S,R219I, R219A, R219M, R219T, R219E, R219Y, R219D, R219C, R219L, V222F,V222C, V222E, W223Y, A225K, N226Q, N226C, T227S, T227M, T227C, T227L,L228A, L228F, N229S, L230S, L230A, L230T, L230V, D231G, F233A, F233L,V238C, V238W, I241L, K242C, K242Y, K242P, K242W, K242G, K242L, K242R,K242S, K242E, K242A, K242Q, F245C, F245V, F245D, F245G, M246L, M246F,W249L, L250T, L250A, H252E, H252W, H252C, R254Q, R254S, G255E, G255L,T257V, G258V, G258C, G258I, G258L, G258P, K259V, K259Q, K259I, K259L,N260E, N260D, N260C, N260W, L261G, L261D, F262Q, F262L, F262E, F262R,F262P, F262V, A263L, A263C, Y267V, Y267L, W268V, K269L, K269W, N270D,N270A, N270M, D271V, D271C, G273W, G273R, G273V, A274S, A274D, A274E,Y278G, Y278S, L279V, L279G, L279Q, S280C, K281L, K281W, K281G, K281A,T282R, T282I, N283V, N283A, W284E, W284C, W284V, W284A, T285E, T285F,T285S, M286D, M286L, S287V, S287Q, S287T, S287E, S287P, S287L, S287W,S287C, A288L, A288Y, V291T, P292A, P292Q, P292R, P292L, L293E, L293T,L293G, H294G, H294A, Y295F, Y295N, N296A, N296Y, N296M, N296F, L297S,L297W, Q299I Q299S, Q299L, Q299R, A300V, S301R, S301Q, S303I, S303E,S304P, G305S, N306V, L312F, L312V, L313S, N314F, N314D, G315I, G315V,T316S, V318C, V318E, V318A, V318L, Q319L, Q319I, Q319A, R320L, R320V,R320C, H321E, H321V, H321C, H321T, P322E, P322C, A325V, A325S, P336M,P336E, P336D, P336R, P336L, G337T, G337C, G337M, G337V, E341C, E341D,E341I, S342G, S342T, S342A, V344S, V344L, Q345K, Q345C, Q345V, Q345I,Q345L, G346H, G346A, G346L, G346N, G346Q, G346V, G346R, W347L, F348Q,F348S, F348C, K349G, K349A, K349Y, K349L, K349S, P350L, 350G, P350S,P350Q, L351M, L351S, A352S, A352C, L357C, L357T, L357S, T358V, T358F,T358S, Q361P, Q361R, Q361A, Q361E, Q361V, G362Y, G362R, G362C, G362N,G362M, G362V, Y363Q, Y363I, Y363A, Y363L, Q365D, V366A, Y368M, Y368T,Y368I, Y368L, Y368A, Y368S, Y368Q, Y368V, Y368H, G369K, G369S, G369A,G369E, G369R, G369I, D370A, D370T, Y372S, Y372I, Y372L, Y372G, G373C,G373S, I374Y, P375L, P375A, P375T, S376P, S376W, S376R, S376L, D377C,D377L, D377V, V379F, V379P, V379L, P380L, P380G, P380E, P380A, P380I,P380V, S381N, S381L, S381A, S381I, S381V, Y382I, Y382A, Y382E, Y382V,Y382L, R383G, R383S, R383P, Q384L, Q384H, Q384R, Q384Y, I386T, D387H,D387V, D387Q, D387L, P388G, P388T, P388M, P388W, P388V, L389S, L389H,L390I, L390G, K391Y, K391A, A392I, Q394S, Q394R, Q395N, Q395L, Q395E,Q395K, Q395R, Y396L, A397C, A397G, R400L, R400C, R400K, R400S, R400M,H402V, H402E, H402M, H402A, H402S, H402P, H402Y, H402L, F405C, F405V,F405I, F405P, F405W, F405G, D406E, H407C, H407D, W408R, W408P, W408C,W408Y, W408G, W408D, V410I, V410Q, T414V, T414A, R415F, R415L, E416C,E416V, N418L, N418A, N418C, N418M, N418G, A419E, A419C, A419M, H421V,H421C, H421A, H421R, G425A, A427V, T428G, I429V, 1429A, M430R, S431A,G435E, G435N, G436M, G436E, G436L, G436T, S437Q, S437L, W439C, W439H,W439E, W439R, W439N, M440L, V442I, R444A, R444S, R444L, R444V, R444P,Q445L, Q445S, Q445C, Q445E, A447G, A447Q, A447E, A447T, G448S, H452A,H452T, M454R, M454L, M454V, T455S, G456V, G456R, G456Y, N457R, S459V,S459C, S459P, S459M, S459T, S459E, S459Q, G460S, G460D, G460E, T463D,T463H, N465Q, N465L, N465R, D467V, D467C, W469E, W469T, H471Q, F473Y,F473C, F473R, N475T, N475H, G476K, G477Q, G477F, G477K, G477L, V481Q,and V481S, wherein numbering is according to the amino acid sequence setforth in SEQ ID NO: 3, and wherein the one or more substitutionsprovides a variant having an IF≥1.2 for a measure of specific activityin Model A detergent composition, and optionally, an IF≥1.0 for measureof stability.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; H1V, H2T, G7F, G7V, G7E, G7E,G7D, I9M, I9L, Q11L, Q11V, W15G, W15S, N19G, N19S, Q22C, Q22A, W24F,N25V, R26L, H28E, N29G, N30S, A31S, L34Y, K35L, I39L, T40A, A41Q, 144T,W48C, W48I, W48Y, W48F, S52M, S52V, Q53V, Q53L, V56L, G59E, T74V, T74L,V75T, I89A, R90L, S91A, S91G, L92C, N95E, N95S, N95G, I97V, Q98V, Q98L,Y100W, K108V, F113M, F113V, F113E, F113Y, R116Q, V117G, N126G, N126A,N128S, N128E, Q129E, G133A, G133D, A139S, W140H, T141S, T141C, T141G,F143M, F143L, F145A, P146L, Q151L, K156F, K156Q, K156L, K156S, K156G,R158S, T165I, D166S, Q169L, S170A, S170V, R171A, Q172L, Q172E, L173I,A174V, N175G, K179L, R181A, R181G, K185G, A186S, A186D, T193S, Y203W,Y203L, D209S, D209M, D209V, H210N, E212G, E212I, V213W, N215C, L217S,R219L, V222E, N226C, T227L, L228F, K242A, F245G, H252C, T257V, G258P,K259L, N260W, K269W, L279Q, K281A, M286L, P292L, L293T, L293G, Y295F,L297W, Q299S, Q299L, Q299R, N306V, L312F, L312V, G315V, T316S, V318L,R320V, R320C, H321T, P336L, G337V, S342A, V344L, Q345L, G346R, W347L,P350S, P350Q, Q361V, Y363L, Y368H, I374Y, S376R, S376L, V379L, P380V,S381V, S381N, D387Q, D387L, A392I, Q395E, Q395K, Q395R, H402L, F405G,T414A, E416V, N418M, N418G, A419M, S431A, G435N, W439E, W439R, W439N,G456Y, S459E, S459Q, N465L, N465R, and D467C, wherein numbering isaccording to the amino acid sequence set forth in SEQ ID NO: 3, andwherein the one or more substitutions provides a variant having anIF≥1.2 for a measure of specific activity in Model A detergentcomposition and an IF≥1.0 for a measure of stability.

In one embodiment, the variant comprises a substitution at one or morepositions selected from the group consisting of; H1, H2, G4, T5, G7,Q11, F13, E14, W15, N16, P18, N19, D20, Q22, N25, R26, N29, N30, A31,Q32, L34, A41, P46, W48, G50, T51, S52, Q53, D55, V56, G59, A60, Y61,Y64, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, K82, A83, R87,I89, R90, S91, L92, K93, N95, Q98, V99, Y100, H107, K108, G109, D112,F113, E115, R116, V117, A119, V120, E121, V122, N123, N126, R127, N128,Q129, E130, V131, S132, G133, T134, Y135, I137, A139, W140, T141, G142,F143, N144, F145, P146, G147, G149, N150, Q151, S153, F155, K156, W157,R158, W159, Y160, F162, D163, G164, T165, D166, W167, D168, Q169, S170,R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184,K185, A186, W187, D188, W189, V191, T193, E194, N197, D199, Y200, M202,Y203, A204, D205, V206, D209, H210, P211, E212, N215, L217, R219, W223,N226, V238, I241, K242, F245, G258, K259, N260, F262, A263, K269, N270,D271, G273, A274, Y278, L279, K281, W284, T285, S287, P292, N296, L297,Q299, S301, N306, L312, G315, V318, Q319, R320, H321, P322, A325, P336,G337, E341, S342, V344, G346, F348, K349, P350, L351, Q361, G362, Y363,Y368, G369, D370, Y372, G373, S376, D377, V379, P380, S381, Y382, Q384,D387, Q394, Q395, A397, R400, H402, H407, W408, V410, R415, N418, H421,S431, G435, G436, W439, M440, Q445, A447, M454, G456, N465, D467, N475,G477, and V481; wherein the positions correspond to amino acid positionsin the amino acid sequence set forth in SEQ ID NO: 3, which one or moresubstitutions provides an improvement factor of ≥1.4 for a measure ofspecific activity in Model A detergent composition, and optionally, animprovement factor of ≥1.0 for a measure of stability.

In a particular embodiment, the variant comprises one or moresubstitutions selected from the group consisting of; H1V, H1G, H1S, H1C,H2E, H2P, H2G, G4D, G4R, T5E, T5V, G7F, G7V, G7E, G7E, G7D, G7R, Q11V,F13Y, F13I, E14L, E14M, E14S, W15G, W15S, W15F, N16G, N16Q, P18L, N19G,N19S, N19Y, N19T, N19V, D20R, Q22C, Q22A, Q22S, Q22M, Q22T, Q22E, N25V,N25S, N25G, N25E, N25E, R26L, R26G, R26D, R26M, R26I, R26S, R26W, R26A,R26F, N29G, N29L, N30L, N30T, N30E, A31S, A31V, Q32D, Q32G, Q32L, Q32A,L34Y, A41Q, A41D, P46G, W48C, W48I, W48M, W48P, W48Q, W48Y, G50P, G50S,G50N, T51G, T51P, T51L, T51C, S52M, S52V, S52I, S52L, Q53V, Q53L, Q53G,D55V, D55I, V56L, V56M, G59E, G59A, A60S, A60W, A60L, A60G, A60T, Y61F,Y64W, Y64S, Y64V, Y64I, Y64H, Y64M, Y64C, Y64R, Y64G, Y64L, Y64A, F69W,N70G, N70A, N70T, N70S, N70E, Q71N, Q71E, Q71D, Q71I, Q71V, K72Y, K72R,G73V, G73L, G73E, T74V, T74L, V75G, R76G, R76A, T77S, K78L, K78T, K78A,K82A, A83E, R87A, R87E, I89H, I89P, I89A, R90L, R90G, S91E, S91A, S91G,L92C, K93W, K93G, K93L, N95W, N95C, N95E, N95S, N95G, Q98C, Q98M, Q98T,Q98L, V99S, V99T, Y100V, Y100F, Y100W, H107Q, K108V, G109A, G109S,D112E, F113L, F113M, F113V, F113E, F113C, F113D, E115A, R116N, R116L,R116I, R116D, R116T, R116G, R116E, R116S, R116C, R116V, R116Q, R116F,V117C, V117L, V117S, V117W, V117A, V117G, V117E, A119Y, V120L, E121P,E121G, E121V, E121M, E121D, V122E, V122P, V122I, N123A, N123E, N123W,N123T, N123P, N126E, N126G, N126A, R127G, R127S, R127C, R127W, R127L,R127V, R127F, R127Y, N128C, N128S, N128E, Q129C, Q129E, E130C, V131L,V131E, S132A, S132H, S132P, S132C, S132E, S132D, G133V, G133I, G133E,G133A, G133D, T134V, T134D, T134E, Y135L, Y135V, Y135S, Y135R, Y135T,Y135W, Y135K, Y135G, Y135P, Y135M, Y135D, Y135E, I137A, I137S, I137G,A139M, A139S, W140Y, W140S, W140A, W140H, T141M, T141S, T141C, T141G,G142D, G142E, F143M, F143L, N144C, F145C, F145S, F145A, P146S, P146V,P146C, P146L, G147V, G147P, G147D, G147S, G147I, G147L, G147C, G149C,N150C, N150D, N150E, Q151T, Q151C, Q151L, S153C, F155H, F155T, F155L,K156Y, K156V, K156A, K156D, K156W, K156P, K156E, K156C, K156F, K156Q,K156L, K156S, K156G, W157L, R158M, R158A, R158G, R158Q, R158W, R158L,R158T, R158V, R158E, R158Y, R158S, W159N, W159G, W159T, W159C, W159E,W159D, Y160Q, Y160M, Y160T, Y160A, Y160S, Y160V, Y160G, Y160I, Y160L,Y160E, F162I, D163K, D163A, D163R, D163L, G164S, G164T, G164C, T165L,T165M, T165S, T165G, T165I, D166T, D166N, D166S, W167Y, W167G, W167D,D168G, Q169A, Q169L, S170A, S170V, R171F, R171S, R171K, R171V, R171A,Q172S, Q172E, L173G, L173N, L173M, L173S, L173Y, L173V, L173I, A174G,A174V, N175V, N175I, N175G, R176E, R176W, R176G, R176M, I177L, I177S,I177G, Y178V, Y178R, Y178K, K179C, K179E, K179S, K179W, K179A, K179G,K179T, K179F, K179L, F180L, R181K, R181P, R181S, R181V, R181Q, R181E,R181A, R181G, G184R, G184V, K185A, K185D, K185N, K185V, K185G, A186C,A186S, A186D, W187G, D188Q, D188G, D188R, D188M, D188L, D188V, W189R,W189A, W189G, W189C, V191M, V191A, T193C, T193P, E194Q, N197C, N197D,D199G, D199S, D199A, Y200W, Y200F, Y200C, Y200V, Y200T, Y200L, Y200A,Y200G, Y200S, M202T, Y203F, Y203G, Y203C, Y203I, Y203Q, Y203H, Y203T,Y203M, Y203S, Y203V, Y203W, Y203L, A204P, A204R, A204C, A204H, A204W,A204F, A204L, A204Y, A204D, A204G, D205C, V206P, D209V, H210V, H210A,H210W, H210L, H210G, H210D, H210E, P211C, E212M, E212D, E212I, N215D,N215V, N215C, L217P, L217T, L217G, L217S, R219H, R219W, R219S, R219I,R219A, R219M, R219T, R219E, R219Y, R219D, R219C, R219L, W223Y, N226Q,L230V, V238C, V238W, I241L, K242C, K242Y, K242P, K242W, K242G, K242L,K242R, K242S, K242E, K242A, K242Q, F245C, F245V, F245D, F245G, G258L,K259I, N260D, N260C, F262Q, F262L, F262E, F262R, F262P, F262V, A263L,A263C, K269L, K269W, N270D, N270A, N270M, D271C, G273V, A274S, A274D,A274E, Y278S, L279G, K281G, W284V, W284A, T285F, T285S, S287V, S287Q,S287T, S287E, S287P, S287L, S287W, S287C, P292R, N296Y, N296M, N296F,L297W, Q299I, S301Q, N306V, L312F, L312V, G315V, V318E, V318A, Q319A,R320C, H321E, H321V, H321C, P322E, P322C, A325V, A325S, P336E, P336D,P336R, P336L, G337M, G337V, E341D, E341I, S342A, V344L, G346V, F348Q,F348S, F348C, K349G, K349A, K349Y, K349L, K349S, P350L, P350G, P350S,P350Q, L351S, Q361A, Q361E, G362Y, G362R, G362C, G362N, G362M, G362V,Y363Q, Y363I, Y363A, Y368I, Y368L, Y368A, Y368S, Y368Q, Y368V, Y368H,G369K, G369S, G369A, G369E, G369R, G369I, D370T, Y372L, Y372G, G373S,S376L, D377L, D377V, V379P, P380I, S381L, S381A, S381I, S381V, Y382A,Y382E, Y382V, Y382L, Q384R, Q384Y, D387V, D387Q, D387L, Q394S, Q394R,Q395R, A397G, R400L, R400C, R400K, R400S, R400M, H402S, H402P, H402Y,H407D, W408R, W408P, W408C, W408Y, W408G, W408D, V410Q, R415F, R415L,N418A, N418C, N418M, N418G, H421C, H421A, H421R, S431A, G435E, G435N,G436T, W439R, W439N, M440L, R444V, R444P, Q445C, Q445E, A447E, A447T,M454V, G456R, S459T, N465Q, N465L, N465R, D467V, N475H, G477Q, G477F,G477K, G477L, and V481S, wherein the numbering is according to the aminoacid sequence set forth in SEQ ID NO: 3, and wherein the one or moresubstitutions provides a variant having an IF≥1.4 for a measure ofspecific activity in Model A detergent composition, and optionally, anIF≥1.0 for a measure of stability.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; H1V, G7F, G7V, G7E, G7E, G7D,Q11V, W15G, W15S, N19G, N19S, Q22C, Q22A, N25V, R26L, N29G, A31S, L34Y,A41Q, W48C, W48I, W48Y, S52M, S52V, Q53V, Q53L, V56L, G59E, T74V, T74L,I89A, R90L, S91A, S91G, L92C, N95E, N95S, N95G, Q98L, Y100W, K108V,F113M, F113V, F113E, R116Q, V117G, N126G, N126A, N128S, N128E, Q129E,G133A, G133D, A139S, W140H, T141S, T141C, T141G, F143M, F143L, F145A,P146L, Q151L, K156F, K156Q, K156L, K156S, K156G, R158S, T165I, D166S,Q169L, S170A, S170V, R171A, Q172E, L173I, A174V, N175G, K179L, R181A,R181G, K185G, A186S, A186D, Y203W, Y203L, D209V, E212I, N215C, L217S,R219L, K242A, F245G, K269W, L297W, N306V, L312F, L312V, G315V, R320C,P336L, G337V, W439R, W439N, S342A, V344L, P350S, P350Q, Y368H, S376L,S381V, D387Q, D387L, Q395R, N418M, N418G, S431A, G435N, N465L, andN465R, wherein the numbering is according to the amino acid sequence setforth in SEQ ID NO: 3, and wherein the one or more substitutionsprovides a variant having an IF≥1.4 for a measure of specific activityin Model A detergent composition, and an IF≥1.0 for a measure ofstability.

In one embodiment, the variant comprises a substitution at one or morepositions selected from the group consisting of; H1, G7, F13, E14, W15,N16, N19, D20, Q22, R26, N29, N30, A31, Q32, P46, W48, G50, T51, S52,Q53, D55, G59, A60, Y64, N70, Q71, K72, G73, T74, V75, K78, R90, Y100,H107, K108, G109, D112, F113, R116, V117, A119, E121, N123, N126, R127,N128, E130, V131, S132, G133, T134, Y135, I137, A139, W140, T141, G142,N144, F145, P146, G147, G149, N150, Q151, F155, K156, R158, W159, Y160,D163, G164, T165, D166, W167, S170, R171, Q172, L173, R176, Y178, K179,R181, K185, A186, W187, D188, W189, V191, E194, D199, Y200, M202, Y203,A204, D205, H210, N215, L217, R219, W223, V238, K242, F245, G258, F262,K269, N270, A274, T285, S287, N296, L312, V318, H321, P322, A325, G337,E341, S342, K349, P350, Q361, G362, Y368, G369, D377, S381, Q384, D387,R400, W408, R415, N418, G435, Q445, M454, G477, and V481; wherein thepositions correspond to amino acid positions in the amino acid sequenceset forth in SEQ ID NO: 3, which one or more substitutions provides animprovement factor of ≥1.6 for a measure of specific activity in Model Adetergent composition, and optionally, an improvement factor of ≥1.0 fora measure of stability.

In a particular embodiment, the variant comprises one or moresubstitutions selected from the group consisting of; H1C, G7V, G7E, G7E,G7D, F13I, E14L, E14M, E14S, W15S, W15F, N16Q, N19G, N19S, N19V, D20R,Q22A, R26L, R26W, R26A, R26F, N29G, N29L, N30E, A31S, Q32L, Q32A, P46G,W48Y, G50N, T51G, T51P, T51L, T51C, S52V, S52I, S52L, Q53V, Q53L, D55V,D55I, G59A, A60L, A60G, Y64V, Y64I, Y64H, Y64M, Y64C, Y64R, Y64G, Y64L,Y64A, N70S, N70E, Q71I, Q71V, K72R, G73L, G73E, T74V, T74L, V75G, K78T,K78A, R90L, R90G, Y100F, H107Q, K108V, G109A, G109S, D112E, F113E,F113C, F113D, R116I, R116D, R116T, R116G, R116E, R116S, R116C, R116V,R116Q, R116F, V117S, V117W, V117A, V117G, V117E, A119Y, E121G, E121V,E121M, E121D, N123P, N126G, N126A, R127C, R127W, R127L, R127V, R127F,R127Y, N128C, N128E, E130C, V131E, S132H, S132P, S132C, S132E, S132D,G133E, G133D, T134D, T134E, Y135T, Y135W, Y135K, Y135G, Y135P, Y135M,Y135D, I137S, I137G, A139S, W140Y, W140S, W140A, W140H, T141C, T141G,G142E, N144C, F145S, F145A, P146C, P146L, G147I, G147L, G147C, G149C,N150D, N150E, Q151T, Q151C, Q151L, F155L, K156V, K156A, K156D, K156W,K156P, K156E, K156C, K156F, K156Q, K156L, K156S, K156G, R158A, R158G,R158Q, R158W, R158L, R158T, R158V, R158E, R158Y, R158S, W159G, W159T,W159C, W159E, W159D, Y160S, Y160V, Y160G, Y160I, Y160L, Y160E, D163L,G164T, G164C, T165G, T165I, D166T, D166N, D166S, W167D, Q169A, Q169L,S170V, R171V, R171A, Q172S, L173V, R176M, Y178R, Y178K, K179C, K179E,K179S, K179W, K179A, K179G, K179T, K179F, R181V, R181Q, R181E, R181G,K185A, K185D, K185N, K185V, K185G, A186D, W187G, D188M, D188L, D188V,W189R, W189A, W189G, W189C, V191M, V191A, E194Q, D199S, D199A, Y200W,Y200F, Y200C, Y200V, Y200T, Y200L, Y200A, Y200G, Y200S, M202T, Y203I,Y203Q, Y203H, Y203T, Y203M, Y203S, Y203V, Y203W, Y203L, A204H, A204W,A204F, A204L, A204Y, A204D, A204G, D205C, H210W, H210L, H210G, H210D,H210E, N215D, N215V, N215C, L217S, R219Y, R219D, R219C, W223Y, V238C,V238W, K242W, K242G, K242L, K242R, K242S, K242E, K242Q, F245C, F245V,F245D, F245G, G258L, F262E, F262R, F262P, F262V, K269L, N270D, N270A,N270M, A274S, A274D, A274E, T285F, T285S, S287W, S287C, N296M, N296F,L312F, L312V, V318A, H321V, H321C, P322C, A325S, G337M, G337V, E341I,S342A, K349A, K349Y, K349L, K349S, P350G, P350Q, Q361E, G362R, G362C,G362N, G362M, G362V, Y368L, Y368A, Y368S, Y368Q, Y368V, G369K, G369S,G369A, G369E, G369R, G369I, D377V, S381L, S381A, S381I, Q384R, Q384Y,D387L, R400M, W408Y, W408G, W408D, R415L, N418G, G435E, G435N, Q445E,M454V, G477Q, G477F, G477K, G477L, and V481S, wherein the positionscorrespond to amino acid positions in the amino acid sequence set forthin SEQ ID NO: 3, and wherein the one or more substitutions provides avariant having an IF≥1.6 for a measure of specific activity in Model Adetergent composition, and optionally, an IF≥1.0 for a measure ofstability.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; G7V, G7E, G7E, G7D, W15S, N19G,N19S, Q22A, R26L, N29G, A31S, W48Y, S52V, Q53V, Q53L, T74V, T74L, R90L,K108V, F113E, R116Q, V117G, N126G, N126A, N128E, G133D, A139S, W140H,T141C, T141G, F145A, P146L, Q151L, K156F, K156Q, K156L, K156S, K156G,R158S, T165I, D166S, Q169L, S170V, R171A, R181G, K185G, A186D, Y203W,Y203L, N215C, L217S, F245G, L312F, L312V, G337V, S342A, P350Q, D387L,N418G, and G435N, wherein the positions correspond to amino acidpositions in the amino acid sequence set forth in SEQ ID NO: 3, andwherein the one or more substitutions provides a variant having anIF≥1.6 for a measure of specific activity in Model A detergentcomposition, and an IF≥1.0 for a measure of stability.

In another embodiment, the variant comprises a substitution at one ormore positions selected from the group consisting of: H1, H2, D3, G4,T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, P18, N19, Q22, W24,N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38,I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, G50, T51, S52, Q53,N54, D55, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73,T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89,R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108,G109, D112, F113, E115, R116, V117, A119, V120, E121, V122, N123, Q125,N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, I137, E138,A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151,S153, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165,D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177,Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191,D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204,D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218,R219, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233,V238, I241, K242, F245, M246, W249, L250, H252, V253, R254, G255, Q256,T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270,D271, G273, A274, E276, Y278, L279, S280, K281, T282, N283, W284, T285,M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299,A300, S301, N302, S303, S304, G305, N306, Y307, M309, L312, L313, N314,G315, T316, V318, Q319, R320, H321, P322, A325, V326, T327, V329, N331,P336, G337, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351,A352, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, Y368, G369,D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383,Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397,R400, H402, D403, F405, D406, H407, W408, V410, I411, T414, R415, E416,N418, A419, S420, H421, S424, G425, A427, T428, I429, M430, S431, G435,G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448,V450, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465,Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; whereinthe positions correspond to amino acid positions in the amino acidsequence as set forth in SEQ ID NO: 3, and wherein the one or moresubstitutions provides a variant having an IF of ≥1.2 for a measure ofspecific activity in Model X detergent composition and optionally an IFof ≥1.0 for a measure of stability.

In one embodiment, the variant comprises one or more substitutionsselected form the group consisting of; H1V, H1G, H1S, H1C, H2L, H2T,H2M, H2E, H2P, H2G, D3C, D3A, G4Q, G4D, T5L, T5Q, T5G, T5E, T5V, G7F,G7R, G7V, G7E, G7E, G7D, T8A, T8S, I9C, I9M, I9L, M10L, Q11C, Q11L,Q11V, Y12V, Y12A, Y12F, F13Y, F13I, E14L, E14M, E14S, W15G, W15F, W15S,N16G, N16Q, P18L, N19Y, N19T, N19G, N19S, N19V, Q22N, Q22C, Q22S, Q22M,Q22T, Q22E, Q22A, W24H, W24F, N25V, N25S, N25G, N25E, N25E, R26G, R26D,R26M, R26I, R26S, R26L, R26W, R26A, R26F, L27A, L27M, H28A, H28E, H28I,N29C, N29G, N29L, N30S, N30H, N30L, N30T, N30E, A31W, A31V, A31S, Q32D,Q32G, Q32L, Q32A, N33F, N33A, N33R, L34F, L34Y, K35L, K35E, K35W, K35Q,N36R, N36Q, N36A, N36E, A37R, A37G, A37T, A37S, G38N, 139L, I39M, T40Q,T40D, T40A, T40E, A41E, A41N, A41M, A41Q, A41D, I42V, I42A, W43R, I44T,P45A, P46G, A47S, A47G, W48G, W48L, W48C, W48I, W48M, W48P, W48Q, W48Y,W48F, G50P, G50S, G50N, T51S, T51G, T51P, T51L, T51C, S52M, S52V, S52I,S52L, Q53G, Q53V, Q53L, N54G, N54K, N54V, D55V, D55I, V56L, V56M, G59E,G59A, A60S, A60W, A60L, A60G, A60T, Y61F, L63V, L63P, L63I, L63H, L63K,Y64W, Y64S, Y64V, Y64I, Y64H, Y64M, Y64C, Y64R, Y64G, Y64L, Y64A, L66M,E68D, F69H, F69W, N70G, N70A, N70T, N70S, N70E, Q71N, Q71E, Q71D, Q71I,Q71V, K72Y, K72R, G73V, G73L, G73E, T74V, T74L, V75T, V75G, R76S, R76G,R76A, T77A, T77S, K78L, K78T, K78A, T81G, T81W, T81L, K82A, A83G, A83E,E84F, E84D, L85V, E86L, E86D, R87M, R87A, R87E, A88S, A88G, A88V, I89A,I89H, I89P, R90L, R90G, S91A, S91E, S91G, L92C, K93W, K93G, K93L, N95L,N95E, N95W, N95C, N95S, N95G, G96Q, 197M, I97V, Q98V, Q98L, Q98C, Q98M,Q98T, V99I, V99L, V99S, V99T, Y100W, Y100V, Y100F, V103G, H107Q, K108V,G109A, G109S, D112E, F113M, F113V, F113L, F113C, F113E, F113D, F113Y,E115C, E115A, R116N, R116L, R116I, R116Q, R116D, R116T, R116G, R116E,R116S, R116C, R116V, R116F, V117C, V117L, V117S, V117W, V117A, V117G,V117E, A119Y, V120I, V120D, V120L, E121P, E121G, E121V, E121M, E121D,V122E, V122P, V122I, N123G, N123A, N123E, N123W, N123T, N123P, Q125E,Q125T, N126E, N126G, N126A, R127G, R127S, R127C, R127W, R127L, R127V,R127F, R127Y, N128S, N128E, N128C, Q129L, Q129C, Q129E, E130C, V131L,V131E, S132A, S132H, S132P, S132C, S132E, S132D, A132N, G133S, G133T,G133L, G133A, G133V, G133I, G133E, G133D, T134V, T134D, T134E, Y135L,Y135V, Y135S, Y135R, Y135T, Y135W, Y135K, Y135G, Y135P, Y135M, Y135D,Y135H, Y135E, Y135E, I137A, I137S, I137G, E138A, A139M, A139S, W140Y,W140S, W140H, W140A, T141S, T141M, T141C, T141G, G142D, G142E, F143M,F143L, N144C, F145C, F145S, F145A, P146S, P146V, P146L, P146C, G147V,G147P, G147D, G147S, G147I, G147L, G147C, G149C, N150C, N150D, N150E,Q151L, Q151T, Q151C, S153C, F155H, F155T, F155L, K156Y, K156V, K156A,K156F, K156D, K156Q, K156L, K156S, K156W, K156P, K156E, K156C, K156G,W157L, R158M, R158A, R158G, R158Q, R158S, R158W, R158L, R158T, R158V,R158E, R158Y, W159N, W159G, W159T, W159C, W159E, W159D, W159H, Y160Q,Y160M, Y160T, Y160A, Y160S, Y160V, Y160G, Y160I, Y160L, Y160E, H161C,H161N, F162M, F162L, F162Y, F162I, D163S, D163K, D163A, D163R, D163L,G164S, G164T, G164C, T165L, T165M, T165S, T165G, T165I, D166T, D166S,D166N, W167Y, W167G, W167D, D168M, D168S, D168G, Q169L, S170A, S170V,R171F, R171S, R171K, R171A, R171V, Q172L, Q172E, Q172S, L173I, L173G,L173N, L173M, L173S, L173Y, L173V, A174V, A174G, N175L, N175V, N175I,N175G, R176E, R176W, R176G, R176M, I177L, I177S, I177G, Y178V, Y178R,Y178K, K179L, K179C, K179E, K179S, K179W, K179A, K179G, K179T, K179F,F180A, F180L, R181K, R181P, R181S, R181A, R181V, R181Q, R181G, R181E,G184L, G184R, G184V, G184D, K185A, K185D, K185N, K185G, K185V, A186S,A186C, A186D, W187G, D188Q, D188G, D188R, D188M, D188L, D188V, W189R,W189A, W189G, W189C, E190D, E190N, E190A, V191M, V191A, D192E, D192Q,D192S, T193S, T193K, T193V, T193C, T193P, E194S, E194D, E194L, E194Q,N195R, N195A, G196A, G196V, N197G, N197V, N197Y, N197C, N197D, Y198W,D199G, D199S, D199A, Y200W, Y200F, Y200C, Y200V, Y200T, Y200L, Y200A,Y200G, Y200S, M202T, Y203F, Y203G, Y203C, Y203I, Y203Q, Y203H, Y203W,Y203T, Y203L, Y203M, Y203S, Y203V, A204P, A204R, A204C, A204H, A204W,A204F, A204L, A204Y, A204D, A204G, D205C, V206H, V206Q, V206P, M208R,M208P, M208L, M208C, D209C, D209S, D209M, D209E, D209H, D209F, D209Y,D209V, H210F, H210V, H210A, H210W, H210L, H210G, H210D, H210E, H210N,P211G, P211W, P211C, E212G, E212F, E212M, E212D, E212I, V213C, V213M,V213T, V213G, V213W, I214P, N215D, N215C, N215V, L217V, L217C, L217P,L217T, L217G, L217S, N218S, R219L, R219H, R219W, R219S, R219I, R219A,R219M, R219T, R219E, R219Y, R219D, R219C, V222E, V222F, V222C, W223Y,Y224L, A225K, N226C, N226Q, T227S, T227L, T227M, T227C, L228A, L228F,N229T, N229S, L230S, L230A, L230T, L230V, F233A, F233L, V238C, V238W,I241L, K242A, K242C, K242Y, K242P, K242W, K242G, K242L, K242R, K242S,K242E, K242Q, F245C, F245V, F245D, F245G, M246L, M246F, W249L, L250C,L250W, L250T, L250A, H252C, H252E, H252W, V253A, V253L, V253S, V253W,R254Q, R254S, G255C, G255E, G255L, Q256P, T257V, G258P, G258V, G258C,G258I, G258L, K259L, K259V, K259Q, K259I, N260W, N260E, N260D, N260C,L261G, L261D, F262Q, F262L, F262E, F262R, F262P, F262V, A263L, A263C,V264T, V264L, Y267V, Y267L, W268V, K269W, K269L, N270D, N270A, N270M,D271E, D271V, D271C, G273W, G273R, G273V, A274S, A274D, A274E, E276R,Y278G, Y278S, L279Q, L279V, L279G, S280C, K281L, K281A, K281W, K281G,T282G, T282A, T282C, T282R, T282I, N283G, N283E, N283Q, N283V, N283A,W284T, W284I, W284S, W284E, W284C, W284V, W284A, T285E, T285F, T285S,M286L, M286D, S287V, S287Q, S287T, S287E, S287P, S287L, S287W, S287C,A288L, A288Y, V291T, P292A, P292Q, P292L, P292R, L293A, L293E, L293T,L293G, H294G, H294A, Y295H, Y295S, Y295F, N296A, N296Y, N296M, N296F,L297S, L297W, Y298V, Y298P, Q299S, Q299L, Q299R, Q299I, A300V, S301A,S301R, S301Q, N302I, N302T, N302M, S303I, S303E, S304I, S304P, G305V,G305L, G305S, N306V, Y307F, Y307W, M309L, L312F, L312V, L313S, N314Y,N314F, N314D, G315I, G315V, T316K, T316S, V318C, V318L, V318E, V318A,Q319L, Q319I, Q319A, R320V, R320L, R320C, H321T, H321E, H321V, H321C,P322E, P322C, A325V, A325S, V326I, T327V, T327L, T327S, V329A, V329S,N331C, N331V, N331I, P336M, P336E, P336D, P336L, P336R, G337T, G337C,G337V, G337M, W439R, W439N, E341C, E341D, E341I, S342G, S342T, S342A,V344S, V344L, Q345K, Q345C, Q345V, Q345L, Q345I, G346H, G346A, G346R,G346L, G346N, G346Q, G346V, W347L, F348Q, F348S, F348C, K349G, K349A,K349Y, K349L, K349S, P350S, P350L, P350G, P350Q, L351W, L351M, L351S,A352S, A352C, T355V, T355A, L357C, L357T, L357S, T358V, T358F, E360S,E360D, E360C, Q361V, Q361P, Q361R, Q361A, Q361E, G362Y, G362R, G362C,G362N, G362M, G362V, Y363L, Y363Q, Y363I, Y363A, Q365D, V366L, V366A,Y368M, Y368T, Y368H, Y368I, Y368L, Y368A, Y368S, Y368Q, Y368V, G369K,G369S, G369A, G369E, G369R, G369I, D370A, D370T, Y372S, Y372I, Y372L,Y372G, G373C, G373S, I374Y, P375L, P375A, P375T, S376A, S376N, S376P,S376R, S376W, S376L, D377C, D377L, D377V, V379L, V379F, V379P, P380L,P380V, P380G, P380E, P380A, P380I, S381N, S381V, S381L, S381A, S381I,Y382I, Y382A, Y382E, Y382V, Y382L, R383G, R383S, R383P, Q384A, Q384P,Q384L, Q384H, Q384R, Q384Y, I386T, D387H, D387Q, D387V, D387L, P388G,P388T, P388M, P388W, P388V, L389S, L389H, L390V, L390S, L390F, L390I,L390G, K391L, K391Y, K391A, A392I, Q394S, Q394R, Q395W, Q395E, Q395K,Q395N, Q395L, Q395R, Y396T, Y396L, A397C, A397G, R400L, R400C, R400K,R400S, R400M, H402L, H402V, H402E, H402M, H402A, H402S, H402P, H402Y,D403E, F405G, F405C, F405V, F405I, F405P, F405W, D406E, H407C, H407D,W408R, W408P, W408C, W408Y, W408G, W408D, V410I, V410Q, I411A, I411L,I411V, T414A, T414V, R415F, R415L, E416C, E416V, N418L, N418M, N418A,N418C, N418G, A419M, A419E, A419C, S420E, S420T, S420V, H421V, H421C,H421A, H421R, S424C, S424A, G425A, A427V, T428L, T428N, T428A, T428G,I429V, 1429A, M430L, M430R, S431A, G435N, G435E, G436M, G436E, G436L,G436T, S437G, S437Q, S437L, K438T, W439E, W439C, W439H, M440L, Y441N,Y441H, Y441Q, V442I, R444A, R444S, R444L, R444V, R444P, Q445L, Q445S,Q445C, Q445E, K446G, K446Q, A447G, A447Q, A447E, A447T, G448S, V450T,V450A, H452A, H452T, D453E, M454R, M454L, M454V, T455L, T455S, G456Y,G456V, G456R, N457V, N457I, N457G, N457R, S459E, S459Q, S459V, S459C,S459P, S459M, S459T, G460S, G460D, G460E, T463C, T463V, T463L, T463D,T463H, I464V, N465G, N465L, N465R, N465Q, Q466L, Q466C, Q466E, D467C,D467V, W469M, W469E, W469T, H471Q, F473Y, F473C, F473R, N475T, N475H,G476Y, G477Q, G477F, G477K, G477L, V481Q, V481S, W482F, wherein thepositions correspond to amino acid positions in the amino acid sequenceas set forth in SEQ ID NO: 3, and wherein the one or more substitutionsprovides a variant having an IF of ≥1.2 for a measure of specificactivity in Model X detergent compositions, and optionally an IF of ≥1.0for a measure of stability.

In one embodiment, the variant comprises a substitution in one or morepositions selected from the group consisting of; H1, H2, D3, G4, T5, G7,I9, Q11, F13, E14, N16, N19, Q22, W24, N25, R26, N29, A31, Q32, L34,N36, A37, A41, P45, P46, W48, G50, T51, S52, Q53, G59, A60, Y61, L63,Y64, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K82, R87, I89, R90,S91, L92, K93, N95, Q98, V99, Y100, K108, G109, D112, F113, R116, V117,A119, V120, E121, V122, N123, N126, R127, N128, Q129, E130, V131, S132,G133, T134, Y135, I137, A139, W140, T141, G142, F143, N144, F145, P146,G147, G149, N150, Q151, F155, K156, R158, W159, Y160, D163, T165, D166,W167, D168, Q169, R171, Q172, L173, A174, N175, R176, I177, Y178, K179,F180, R181, G184, K185, A186, W187, D188, E194, D199, Y200, M202, Y203,A204, D205, H210, E212, N215, L217, R219, W223, N226, L230, V238, I241,K242, K245, R254, G258, N260, F262, A263, K269, N270, D271, A274, L279,K281, N283, W284, T285, S287, P292, L293, N296, Q299, S301, G305, N306,L312, N314, G315, V318, Q319, R320, H321, P322, A325, G337, W439, E341,S342, V344, Q345, G346, F348, K349, P350, L357, E360, Q361, G362, Y363,Y368, G369, Y372, I374, P375, S376, D377, V379, P380, S381, Y382, Q384,D387, P388, Q394, Q395, A397, R400, H402, W408, V410, R415, N418, H421,G425, S431, G435, G436, W439, R444, Q445, A447, G456, S459, N465, W469,G477, and V481, wherein the positions correspond to amino acid positionsin the amino acid sequence as set forth in SEQ ID NO: 3, and wherein thesubstitution in one or more positions provides a variant having an IF of≥1.4 for a measure of specific activity in Model X detergent compositionand optionally, an IF of at least ≥1.0 for a measure of stability.

In one embodiment, the variant comprises one or more of thesubstitutions selected from the group consisting of; H1G, H1C, H2G, D3A,G4D, T5Q, T5E, T5V, G7F, G7E, G7R, G7V, G7E, G7D, 19M, Q11V, F13I, E14M,N16G, N16Q, N19Y, N19T, N19G, N19S, N19V, Q22N, Q22C, Q22S, Q22M, Q22A,W24F, N25G, N25E, N25E, R26G, R26D, R26I, R26S, R26L, R26W, R26A, R26F,N29G, N29L, A31W, A31S, Q32G, Q32L, Q32A, L34Y, N36E, A37G, A41Q, P45A,P46G, W48G, W48C, W48I, W48M, W48P, W48Q, G50S, G50N, T51G, T51C, S52V,S52I, S52L, Q53L, Q53G, Q53V, G59A, A60S, A60W, A60L, A60G, A60T, Y61F,L63V, L63H, L63K, Y64W, Y64S, Y64V, Y64I, Y64H, Y64M, Y64C, Y64R, Y64G,Y64L, Y64A, F69W, N70A, N70T, N70S, N70E, Q71N, Q71D, Q71I, Q71V, K72Y,K72R, G73V, G73L, G73E, T74L, V75G, R76A, T77S, K82A, R87A, I89A, I89P,R90L, R90G, S91A, S91E, S91G, L92C, K93W, K93G, N95W, N95C, N95G, Q98L,Q98C, Q98M, Q98T, V99S, V99T, Y100W, Y100V, Y100F, K108V, G109A, G109S,D112E, F113M, F113V, F113L, F113C, F113E, F113D, R116N, R116L, R116I,R116Q, R116D, R116T, R116G, R116E, R116S, R116C, R116V, R116F, V117C,V117S, V117W, V117A, V117G, V117E, A119Y, V120D, V120L, E121V, E121M,E121D, V122E, N123G, N123A, N123E, N123W, N123T, N123P, N126E, N126G,N126A, R127G, R127S, R127C, R127W, R127L, R127V, R127F, R127Y, N128C,N128S, N128E, Q129E, E130C, V131L, V131E, S132A, S132H, S132P, S132C,S132E, S132D, G133S, G133V, G133I, G133E, G133D, T134V, T134D, T134E,Y135V, Y135S, Y135R, Y135T, Y135W, Y135K, Y135G, Y135P, Y135M, Y135D,Y135H, Y135E, I137A, I137S, I137G, A139S, W140Y, W140H, T141M, T141C,T141G, T141S, G142D, G142E, F143M, N144C, F145C, F145A, P146C, P146L,G147V, G147P, G147D, G147S, G147I, G147L, G147C, G149C, N150C, N150D,N150E, Q151T, Q151L, F155H, F155L, K156Y, K156V, K156A, K156D, K156Q,K156W, K156P, K156E, K156C, K156F, K156L, K156S, K156G, R158M, R158A,R158G, R158Q, R158S, R158W, R158L, R158T, R158V, R158E, R158Y, W159N,W159G, W159T, W159C, W159E, W159D, Y160Q, Y160T, Y160S, Y160V, Y160G,Y160I, Y160L, Y160E, D163S, D163K, D163L, T165M, T165G, T165I, D166S,W167Y, W167D, D168M, D168S, D168G, Q169L, R171F, R171S, R171K, R171V,R171A, Q172S, Q172E, L173G, L173N, L173M, L173S, L173Y, L173V, A174G,N175L, N175V, N175I, N175G, R176E, R176G, R176M, I177G, Y178V, Y178R,Y178K, K179L, K179C, K179E, K179S, K179W, K179A, K179G, K179T, K179F,F180L, R181K, R181P, R181S, R181V, R181Q, R181E, R181A, R181G, G184V,G184D, K185A, K185D, K185N, K185V, K185G, A186C, A186S, A186D, W187G,D188Q, D188G, D188M, D188L, D188V, E194Q, D199G, D199S, D199A, Y200W,Y200F, Y200C, Y200V, Y200T, Y200L, Y200A, Y200G, Y200S, M202T, Y203I,Y203Q, Y203H, Y203T, Y203M, Y203V, Y203L, A204R, A204H, A204W, A204F,A204L, A204Y, A204D, A204G, D205C, H210F, H210A, H210W, H210L, H210G,H210D, H210E, E212F, E212M, E212D, E212G, E212I, N215D, N215V, N215C,L217C, L217P, R219H, R219I, R219M, R219T, R219E, R219Y, R219D, R219C,W223Y, N226Q, N226C, L230V, V238C, V238W, I241L, K242C, K242Y, K242P,K242W, K242G, K242L, K242R, K242S, K242E, K242A, K242Q, F245C, F245V,F245D, F245G, R254Q, R254S, G258L, N260D, F262Q, F262L, F262E, F262R,F262P, F262V, A263L, A263C, K269L, K269W, N270D, N270A, N270M, D271C,A274S, A274D, A274E, L279Q, L279G, K281W, K281G, N283Q, N283V, N283A,W284C, W284V, W284A, T285F, T285S, S287V, S287Q, S287P, S287L, S287W,S287C, P292R, L293G, N296M, N296F, Q299S, Q299I, S301Q, G305V, G305L,N306V, L312F, L312V, N314F, G315I, G315V, V318A, Q319A, R320L, R320C,H321E, H321V, H321C, P322E, P322C, A325V, A325S, G337C, G337V, G337M,E341D, E341I, S342A, V344L, Q345C, G346Q, G346V, F348Q, F348S, F348C,K349L, K349S, P350S, P350L, P350G, P350Q, L357T, E360C, Q361A, Q361E,G362R, G362C, G362N, G362M, G362V, Y363I, Y363A, Y368I, Y368L, Y368V,G369A, G369R, Y372L, Y372G, I374Y, P375T, S376L, D377V, V379F, V379P,V379L, P380G, P380I, S381N, S381L, S381A, S381I, S381V, Y382V, Q384R,Q384Y, D387L, P388W, Q394S, Q394R, Q395R, A397G, R400M, H402L, H402S,H402P, W408P, W408C, W408Y, V410Q, R415L, N418C, N418G, H421R, G425A,S431A, G435N, G435E, G436M, G436E, G436T, W439H, W439R, W439N, R444V,Q445C, Q445E, A447E, A447T, G456R, S459T, N465R, N465Q, W469M, W469E,G477Q, G477F, G477K, G477L, and V481S, wherein the positions correspondto amino acid positions in the amino acid sequence as set forth in SEQID NO: 3, and wherein the one or more substitution provides a varianthaving an IF of ≥1.4 for a measure of specific activity in Model Xdetergent composition, and optionally an IF of ≥1.0 for a measure ofstability.

In one embodiment, the variant comprises a substitution in one or morepositions selected from the group consisting of; G7, F13, N16, N19, R26,N29, A31, P46, G50, T51, S52, Q53, G59, A60, Y64, N70, Q71, K72, G73,T74, R90, K108, G109, F113, R116, V117, A119, E121, V122, N123, N126,R127, E130, V131, S132, G133, T134, Y135, I137, T141, G142, F143, N144,P146, G147, G149, N150, F155, K156, R158, W159, Y160, D163, T165, W167,D168, Q169, R171, Q172, L173, N175, Y178, K179, R181, K185, A186, W187,D188, E194, Y200, Y203, A204, D205, H210, N215, R219, W223, V238, K242,K245, G258, F262, K269, N270, A274, L279, K281, W284, T285, S287, Q299,L312, H321, A325, G337, E341, S342, F348, K349, P350, Q361, G362, Y368,G369, D377, V379, P380, S381, Q384, V410, N418, G435, Q445, G456, N465,and G477 wherein the positions correspond to amino acid positions in theamino acid sequence set forth in SEQ ID NO: 3, and wherein thesubstitution in one or more positions provides a variant having an IF of≥1.6 for measure of specific activity in Model X detergent composition,and optionally, an IF of ≥1.0 for a measure of stability.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; G7V, G7E, G7D, F13I, N16Q, N19T,N19G, N19S, N19V, R26A, N29G, A31S, P46G, G50N, T51G, T51C, S52I, S52L,Q53L, Q53G, Q53V, G59A, A60S, A60W, A60G, Y64V, Y64M, Y64C, Y64R, Y64G,Y64L, Y64A, N70E, Q71V, K72R, G73L, G73E, T74L, R90G, K108V, G109S,F113V, F113C, F113E, F113D, R116I, R116Q, R116D, R116T, R116G, R116E,R116S, R116C, R116V, R116F, V117A, V117G, V117E, A119Y, E121D, V122E,N123T, N126G, R127W, R127L, R127F, R127Y, E130C, V131L, V131E, S132H,S132P, S132C, S132E, S132D, G133E, G133D, T134V, T134D, T134E, Y135S,Y135T, Y135W, Y135K, Y135G, Y135P, Y135M, Y135D, Y135E, I137S, I137G,T141G, G142D, G142E, N144C, P146C, P146L, G147S, G147L, G149C, N150D,N150E, F155L, K156Y, K156A, K156D, K156Q, K156W, K156P, K156E, K156C,K156F, K156L, K156S, K156G, R158A, R158G, R158Q, R158S, R158W, R158L,R158T, R158V, R158E, R158Y, W159T, W159C, W159E, W159D, Y160S, Y160V,Y160I, Y160L, Y160E, D163L, T165I, W167D, D168G, Q169L, R171V, Q172S,L173V, N175L, N175V, N175I, N175G, Y178K, K179A, K179G, K179T, K179F,R181V, R181Q, R181E, R181G, K185A, K185D, K185N, K185V, K185G, A186S,W187G, D188V, E194Q, Y200C, Y200T, Y200L, Y200A, Y200G, Y200S, Y203M,Y203V, Y203L, A204L, A204D, A204G, D205C, H210F, H210A, H210W, H210L,H210G, H210D, H210E, N215V, R219Y, R219D, R219C, W223Y, V238W, K242R,K242S, K242E, K242A, K242Q, F245D, F245G, G258L, F262Q, F262E, F262R,F262P, F262V, K269L, N270D, N270M, A274E, L279G, K281G, W284A, T285F,S287V, S287W, S287C, Q299I, L312F, L312V, H321V, H321C, A325S, G337V,G337M, E341I, S342A, F348Q, F348S, K349L, K349S, P350L, P350G, Q361E,G362R, G362V, Y368L, Y368V, G369A, D377V, V379P, P380I, S381L, S381A,S381I, Q384Y, V410Q, N418G, G435E, Q445E, G456R, N465R, and G477L,wherein the positions correspond to amino acid positions in the aminoacid sequence as set forth in SEQ ID NO: 3, and wherein the one or moresubstitutions provide a variant having an IF of ≥1.6 for a measure ofspecific activity in Model X detergent composition and optionally an IFof ≥1.0 for a measure of stability.

The variants according to the present invention have further been shownto have an improved stability in liquid detergent compositions. Thus, inone embodiment, variant has an improved stability, such as an improvedstorage stability.

In one embodiment, the variant comprises a substitution in one or morepositions selected from the group consisting of; H1, H2, D3, T5, G7, I9,Q11, E14, W15, N16, V17, N19, Q22, H23, W24, N25, R26, H28, N29, N30,A31, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, I44, A47, W48,K49, T51, S52, Q53, N54, D55, V56, G59, A60, L63, E68, G73, T74, V75,R76, T77, T81, K82, A83, E84, E86, I89, R90, S91, L92, N95, G96, I97,Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114,R116, V117, Q118, V120, V122, N123, P124, N126, N128, Q129, V131, G133,Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G149,Q151, S153, S154, F155, K156, R158, Y160, H161, T165, D166, Q169, S170,R171, Q172, L173, A174, N175, R176, Y178, K179, R181, G184, K185, A186,T193, N195, G196, Y198, Y203, D205, V206, D209, H210, E212, V213, I214,N215, L217, N218, R219, W220, G221, V222, W223, N226, T227, L228, D231,F233, V238, H240, K242, S244, F245, R247, D248, W249, L250, G251, H252,V253, T257, G258, K259, N260, L261, V264, W268, K269, L272, G273, L275,E276, N277, L279, S280, K281, T285, M286, A288, P292, L293, H294, Y295,N296, L297, Q299, A300, N302, S303, S304, G305, N306, R310, N311, L312,L313, G315, T316, V318, Q319, R320, H321, S323, V326, T327, F328, V329,T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, P350,L351, A354, T355, L357, T358, E360, Q361, Y363, Q365, V366, F367, Y368,G369, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383,Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397,Y398, R400, H402, D403, Y404, F405, D406, D409, V410, T414, R415, E416,N418, A419, S420, H421, P422, S424, G425, L426, A427, M430, S431, D432,G435, W439, Y441, Q445, K446, A447, G448, V450, W451, H452, M454, G456,S459, T463, N465, D467, F473, N475, G476, and W482; wherein thepositions correspond to amino acid positions of the amino acid sequenceas set forth in SEQ ID NO: 3, and wherein the one or more substitutionsprovides a variant having an IF of ≥1.0 for a measure of stability.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; H1V, H1L, H2T, D3R, D3Y, D3W,D3S, D3F, T5L, T5F, T5W, G7F, G7V, G7E, G7E, G7D, G7L, G7A, G7S, G7N,I9M, I9L, Q11L, Q11V, Q11M, E14Q, E14F, W15G, W15S, W15I, W15H, N16R,V17D, V17G, V17E, V17Q, V17A, V17T, V17L, N19G, N19S, Q22C, Q22A, Q22Y,H23W, W24F, N25V, N25R, N25L, N25T, R26L, R26E, H28E, H28R, H28M, H28W,H28K, N29G, N29K, N29V, N29W, N29A, N30S, N30R, A31S, A31G, A31R, N33D,L34Y, K35L, N36V, N36P, N36W, N36T, A37E, G38M, G38Y, G38K, 139L, I39V,T40Q, T40A, T40S, A41Q, I42V, I42L, I44T, I44L, I44V, A47G, A47T, W48C,W48I, W48H, W48A, W48Y, W48F, K49Q, T51A, S52M, S52V, S52C, S52F, S52A,S52D, S52K, S52N, S52E, S52T, Q53V, Q53L, Q53M, N54T, N54L, N54I, D55G,V56L, G59E, G59S, A60V, A60F, L63C, L63G, L63A, E68S, E68M, E68C, E68T,E68V, E68Q, E68Y, G73C, T74V, T74L, T74N, V75T, V75E, R76V, T77V, T81G,K82G, A83Q, A83R, A83S, A83F, A83V, E84M, E84W, E86W, E86R, I89A, I89L,R90L, S91A, S91G, S91V, S91T, L92C, L92F, N95E, N95S, N95G, N95D, N95R,G96R, 197V, 197L, Q98V, Q98L, Q98F, Q98W, Q98P, Q98H, V99G, V99E, Y100W,V103A, V103S, V103T, H107M, K108V, G109D, G109F, G110C, A111S, D112V,D112F, D112C, D112K, F113M, F113V, F113E, F113K, F113Y, T114S, T114V,T114L, R116Q, R116K, R116W, V117G, V117I, V117M, Q118V, Q118G, Q118E,Q118F, Q118R, V120C, V120A, V120N, V122L, N123K, N123V, P124R, P124G,N126G, N126A, N126R, N126T, N128S, N128E, N128P, N128G, N128A, Q129E,V131I, G133A, G133D, G133R, Q136E, Q136W, Q136G, I137V, E138A, E138Y,E138T, E138L, A139S, A139G, A139V, A139T, A139P, W140H, T141S, T141C,T141G, G142R, G142A, F143M, F143L, F143I, N144M, N144K, N144G, N144R,F145A, F145N, F145Q, F145T, F145H, P146L, P146M, P146T, G149Y, G149L,G149A, Q151L, Q151I, S153G, S153T, S154M, S154E, S154A, S154V, F155W,K156F, K156Q, K156L, K156S, K156G, K156N, K156T, R158S, Y160W, Y160R,Y160H, H161C, T165I, T165V, T165E, D166S, Q169L, Q169S, Q169V, Q169F,S170A, S170V, S170Q, S170W, S170G, S170F, S170T, S170L, R171A, R171M,Q172L, Q172E, Q172C, Q172R, Q172G, Q172W, L173I, L173A, A174V, A174L,A174M, A174K, N175G, N175S, R176Y, R176M, R176T, R176K, Y178W, K179L,R181A, R181G, R181D, G184D, K185G, A186S, A186D, A186T, A186Q, A186V,A186H, T193S, T193D, T193R, T193G, N195T, N195F, G196P, Y198A, Y198G,Y203W, Y203L, D205R, D205A, D205L, V206A, V206S, V206K, D209S, D209M,D209V, D209Q, D209L, D209I, H210Q, H210N, E212G, E212I, E212Y, E212R,E212C, E212K, V213C, V213W, V213R, V213F, I214V, N215C, N215T, L217S,L217A, L217W, N218S, N218D, N218R, N218L, N218M, R219L, W220L, W220I,W220P, W220E, W220R, W220A, W220S, G221S, G221A, V222E, V222L, W223F,W223L, N226C, N226M, N226E, N226K, N226R, T227L, T227Y, T227R, L228F,L228M, D231P, F233T, F233P, F233M, V238A, H240W, K242A, S244Q, F245G,F245A, R247Y, R247G, R247M, D248G, D248R, W249F, L250C, L250F, L250Y,G251K, G251M, G251Q, H252C, H252T, H252F, H252V, V253A, V253L, T257V,G258P, G258F, G258D, K259L, K259A, K259S, K259D, K259C, N260W, N260S,N260R, N260G, L261T, V264L, W268Y, K269W, K269Q, K269R, L272M, G273E,L275G, L275E, L275A, L275R, L275K, E276L, N277D, L279Q, L279M, S280R,S280E, S280D, K281A, T285W, T285M, T285P, M286L, M286C, A288V, P292L,P292G, L293T, L293G, L293Y, L293Q, H294W, H294Q, H294S, Y295I, Y295A,Y295F, N296S, N296K, N296H, L297W, L297H, Q299S, Q299L, Q299R, Q299A,Q299H, Q299P, A300C, N302S, S303A, S303M, S304G, S304L, S304F, S304A,S304M, G305Q, G305F, G305I, N306V, N306L, N306D, N306S, N306T, R310A,R310T, N311L, N311R, N311C, N311Y, L312F, L312V, L312I, L313M, L313I,G315V, G315T, G315R, G315F, T316S, T316C, T316G, V318L, Q319P, Q319H,Q319D, Q319F, R320V, R320C, R320A, R320S, H321T, H321G, H321R, H321S,S323N, V326G, V326A, V326C, V326T, T327V, T327L, T327Q, T327E, F328M,F328C, V329S, V329C, V329I, T334A, T334S, P336L, P336W, P336S, P336K,G337V, G337F, E338T, E338A, E338H, E341P, E341M, E341F, E341K, E341S,E341T, S342A, V344L, Q345L, Q345W, Q345N, G346R, G346S, W347L, W347R,W347V, W347C, F348P, F348M, P350S, P350Q, P350D, P350E, L351W, L351H,L351Q, A354C, T355L, T355Y, T355F, L357H, T358L, E360G, E360A, E360M,Q361V, Q361G, Q361D, Q361S, Y363L, Y363R, Y363V, Q365M, Q365L, Q365T,Q365C, Q365S, V366I, V366C, F367L, F367S, F367A, Y368H, G369L, Y372A,G373T, G373Q, G373W, G373R, I374Y, I374S, I374M, I374L, P375E, P375S,P375V, P375R, S376R, S376L, S376V, S376I, S376Q, S376F, S376E, S376T,D377S, D377G, D377T, V379L, V379S, P380V, P380Y, S381V, Y382K, Y382R,R383V, Q384S, Q384V, Q384T, Q384G, Q384E, I386L, I386M, I386W, I386V,D387Q, D387L, P388S, P388R, P388K, P388L, L389A, L389V, L389F, L389M,L390V, L390T, L390N, L390H, L390C, K391Q, A392I, A392M, A392T, Q394N,Q394L, Q394T, Q394M, Q395E, Q395K, Q395R, Q395V, Q395S, Q395M, Y396R,Y396W, A397S, Y398S, Y398C, Y398N, Y398K, R400E, R400I, H402L, H402D,H402R, H402T, D403P, D403Q, D403I, Y404K, Y404F, Y404W, F405G, F405A,D406V, D406T, D406M, D406S, D409G, V410L, V410E, T414A, R415A, R415Q,E416V, E416L, E416A, E416Q, N418M, N418G, N418R, N418F, A419M, A419G,A419V, A419L, A419Q, A419R, S420M, H421T, P422Y, P422H, P422D, P422R,S424L, G425V, L426S, L426A, A427S, M430L, M430A, M430T, S431A, S431T,S431F, D432Q, D432P, G435N, G435M, G435I, G435S, W439E, W439A, W439T,W439R, W439N, Y441K, Q445R, K446G, K446T, K446P, A447R, A447S, G448R,V450T, V450Q, V450R, W451L, H452C, H452G, H452Y, H452R, H452V, M454Q,M454S, G456Y, G456K, G456L, S459E, S459Q, S459R, T463M, T463A, T463Q,N465L, N465R, D467C, F473E, F473G, F473T, F473V, N475G, G476F, G476Y,and W482F, wherein the numbering is according to the amino acid sequenceset forth in SEQ ID NO: 3, and wherein the one or more substitutionsprovides a variant having an IF≥1.0 for a measure of stability.

In one embodiment, the variant comprises a substitution at one or morepositions selected from the group consisting of; H1, H2, D3, T5, G7, I9,E14, W15, N16, V17, Q22, H23, W24, N25, R26, H28, N29, N33, N36, A37,G38, I39, T40, A41, I42, I44, W48, S52, Q53, N54, D55, V56, G59, A60,L63, E68, G73, T74, V75, R76, T81, A83, E86, R90, S91, L92, N95, I97,Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114,R116, V117, Q118, V120, V122, N123, P124, N126, N128, Q129, V131, G133,Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G149,Q151, S153, S154, F155, K156, Y160, T165, D166, Q169, S170, R171, Q172,L173, A174, N175, R176, K179, R181, K185, A186, T193, N195, G196, Y198,Y203, D205, V206, D209, H210, E212, V213, I214, N215, L217, N218, W220,G221, W223, N226, T227, L228, D231, F233, V238, H240, K242, S244, R247,D248, L250, G251, H252, T257, G258, K259, N260, L261, K269, L272, G273,L275, E276, N277, L279, K281, T285, M286, A288, P292, L293, H294, Y295,N296, L297, Q299, A300, N302, S303, S304, G305, N306, R310, N311, L312,L313, G315, T316, Q319, R320, H321, T327, T334, P336, E338, E341, S342,V344, G346, W347, F348, P350, L351, A354, T355, T358, E360, Q361, Y363,Q365, F367, Y368, G369, G373, I374, P375, S376, D377, V379, S381, Y382,Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397,Y398, R400, H402, D403, Y404, F405, D406, D409, V410, R415, E416, N418,A419, S420, P422, S424, G425, L426, A427, M430, S431, D432, G435, W439,Y441, Q445, K446, A447, V450, H452, M454, G456, S459, F473, and N475;wherein the positions correspond to amino acid positions in the aminoacid sequence set forth in SEQ ID NO: 3, which one or more substitutionprovides an improvement factor of ≥1.2 for a measure of stability andoptionally, an improvement factor of ≥1.0 for a measure of specificactivity in Model A and/or Model X detergent compositions.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; H1V, H1L, H2T, D3R, D3Y, D3W,D3S, D3F, T5L, T5F, T5W, G7F, G7V, G7E, G7E, G7D, G7L, G7A, I9L, E14Q,W15I, N16R, V17D, V17G, V17E, V17Q, V17A, V17T, V17L, Q22C, Q22A, Q22Y,H23W, W24F, N25V, N25R, N25L, N25T, R26L, R26E, H28E, H28R, H28M, H28W,H28K, N29G, N29V, N29W, N33D, N36V, N36P, N36W, A37E, G38Y, G38K, 139L,I39V, T40Q, T40A, T40S, A41Q, I42L, I44T, W48C, W48I, W48H, W48A, W48Y,W48F, S52M, S52V, S52C, S52F, S52A, S52D, S52K, S52N, S52E, S52T, Q53V,Q53L, Q53M, N54T, N54L, N54I, D55G, V56L, G59E, G59S, A60V, A60F, L63C,L63G, L63A, E68S, E68M, E68C, E68T, E68V, E68Q, E68Y, G73C, T74V, T74L,V75T, V75E, R76V, T81G, A83Q, A83R, A83S, A83F, A83V, E86W, E86R, R90L,S91G, S91T, L92C, N95E, N95S, N95G, N95D, N95R, 197L, Q98V, Q98L, Q98W,Q98P, Q98H, V99G, V99E, Y100W, V103S, V103T, H107M, K108V, G109D, G109F,G110C, A111S, D112F, D112C, D112K, F113M, F113V, F113E, F113K, T114S,T114V, T114L, R116Q, R116K, V117G, V117I, V117M, Q118V, Q118G, Q118E,Q118F, Q118R, V120N, V122L, N123K, N123V, P124R, P124G, N126T, N128S,N128E, N128P, N128G, N128A, Q129E, V131I, G133A, G133D, G133R, Q136E,Q136W, Q136G, I137V, E138A, E138Y, E138T, E138L, A139S, A139G, A139V,A139T, A139P, W140H, T141S, T141C, T141G, G142R, G142A, F143M, F143L,F143I, N144M, N144K, N144G, N144R, F145A, F145N, F145Q, F145T, F145H,P146L, P146M, P146T, G149Y, G149L, G149A, Q151L, S153G, S153T, S154M,S154E, S154A, S154V, F155W, K156F, K156Q, K156L, K156S, K156G, K156N,K156T, Y160H, T165I, D166S, Q169L, Q169S, Q169V, Q169F, S170A, S170V,S170Q, S170W, S170G, S170F, S170T, S170L, R171A, R171M, Q172L, Q172E,Q172C, Q172R, Q172G, Q172W, L173I, L173A, A174V, A174L, A174M, A174K,N175G, N175S, R176Y, R176M, R176T, R176K, K179L, R181A, R181G, R181D,K185G, A186S, A186D, A186T, A186Q, A186V, T193S, T193D, T193R, T193G,N195T, N195F, G196P, Y198G, Y203W, Y203L, D205R, D205A, D205L, V206A,V206S, V206K, D209S, D209M, D209V, D209Q, D209L, D209I, H210N, E212G,E212I, E212Y, E212R, E212C, E212K, V213C, V213W, V213R, V213F, I214V,N215C, N215T, L217S, L217A, L217W, N218S, N218D, N218R, N218L, N218M,W220L, W220I, W220P, W220E, W220R, W220A, W220S, G221A, W223L, N226C,N226M, N226E, N226K, N226R, T227L, T227Y, T227R, L228F, D231P, F233T,F233P, F233M, V238A, H240W, K242A, S244Q, R247Y, R247G, R247M, D248G,D248R, L250C, L250F, L250Y, G251K, G251M, G251Q, H252C, H252T, H252F,H252V, T257V, G258D, K259L, K259A, K259S, K259D, K259C, N260W, N260S,N260R, L261T, K269W, K269Q, K269R, L272M, G273E, L275G, L275E, L275A,L275R, L275K, E276L, N277D, L279Q, L279M, K281A, T285W, T285M, T285P,M286L, M286C, A288V, P292L, P292G, L293T, L293G, L293Y, L293Q, H294W,H294Q, H294S, Y295A, N296S, N296K, N296H, L297W, L297H, Q299S, Q299L,Q299R, Q299A, Q299H, Q299P, A300C, N302S, S303M, S304G, S304L, S304F,S304A, S304M, G305F, G305I, N306V, N306L, N306D, N306S, N306T, R310A,R310T, N311L, N311R, N311C, N311Y, L312F, L312V, L312I, L313M, L313I,G315V, G315T, G315R, G315F, T316S, T316C, T316G, Q319P, Q319H, Q319D,Q319F, R320V, H321T, H321G, H321R, H321S, T327V, T327L, T327E, T334A,T334S, P336L, P336W, P336S, P336K, E338A, E338H, E341P, E341M, E341F,E341K, E341S, S342A, V344L, G346S, W347L, W347R, W347V, W347C, F348M,P350Q, L351W, L351H, L351Q, A354C, T355Y, T355F, T358L, E360G, E360A,E360M, Q361S, Y363L, Y363R, Y363V, Q365S, F367L, F367S, F367A, Y368H,G369L, G373T, G373Q, G373W, G373R, I374Y, I374S, I374M, I374L, P375E,P375S, P375V, P375R, S376R, S376V, S376I, S376Q, S376F, S376E, S376T,D377G, D377T, V379L, V379S, S381V, Y382K, Y382R, Q384S, Q384V, Q384T,Q384G, Q384E, I386W, I386V, D387Q, D387L, P388S, P388R, P388K, P388L,L389A, L389V, L389F, L389M, L390V, L390T, L390N, L390H, L390C, K391Q,A392I, A392M, A392T, Q394N, Q394L, Q394T, Q394M, Q395E, Q395K, Q395R,Y396R, Y396W, A397S, Y398S, Y398C, Y398N, Y398K, R400E, R400I, H402R,H402T, D403I, Y404K, Y404F, Y404W, F405G, F405A, D406S, D409G, V410L,V410E, R415Q, E416V, E416L, E416A, E416Q, N418M, N418G, N418R, N418F,A419M, A419G, A419V, A419L, A419Q, A419R, S420M, P422Y, P422H, P422D,P422R, S424L, G425V, L426S, L426A, A427S, M430L, M430A, M430T, S431A,S431T, S431F, D432Q, D432P, G435N, G435M, G435I, G435S, W439E, W439A,Y441K, Q445R, K446G, K446P, A447S, V450R, H452V, M454Q, M454S, G456Y,S459Q, F473E, F473G, F473T, F473V, and N475G, wherein numbering isaccording to the amino acid sequence set forth in SEQ ID NO: 3, andwherein the one or more substitution provides a variant having an IF of≥1.2 for a measure of stability, and optionally, an IF of ≥1.0 for ameasure of specific activity in Model A and/or Model X detergentcompositions.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; H1V, H2T, T5L, G7F, G7V, G7E,G7E, G7D, 19L, E14F, Q22C, Q22A, W24F, N25V, R26L, H28E, N29G, N29A,I39L, T40Q, T40A, A41Q, 144T, W48C, W48I, W48Y, W48F, T51A, S52M, S52V,Q53V, Q53L, V56L, G59E, T74V, T74L, V75T, R90L, S91G, L92C, N95E, N95S,N95G, Q98V, Q98L, Y100W, K108V, F113M, F113V, F113E, F113Y, R116Q,V117G, N128S, N128E, Q129E, G133A, G133D, E138A, A139S, W140H, T141S,T141C, T141G, F143M, F143L, F145A, P146L, Q151L, K156F, K156Q, K156L,K156S, K156G, T165I, D166S, Q169L, S170A, S170V, R171A, Q172L, Q172E,L173I, A174V, N175G, K179L, R181A, R181G, G184D, K185G, A186S, A186D,A186H, T193S, Y203W, Y203L, D209S, D209M, D209V, H210N, E212G, E212I,V213C, V213W, N215C, L217S, N218S, N226C, T227L, L228F, K242A, L250C,H252C, T257V, K259L, N260W, N260G, K269W, L279Q, K281A, M286L, P292L,L293T, L293G, Y295F, L297W, Q299S, Q299L, Q299R, N306V, L312F, L312V,G315V, T316S, R320V, H321T, S323N, T327V, T327L, P336L, E341T, S342A,V344L, W347L, P350Q, L351W, Y363L, Y368H, I374Y, S376R, V379L, S381V,D387Q, D387L, L390V, A392I, Q395E, Q395K, Q395R, F405G, E416V, N418M,N418G, A419M, M430L, S431A, G435N, W439E, W439R, W439N, K446G, G456Y,S459Q, and G476Y, wherein numbering is according to the amino acidsequence set forth in SEQ ID NO: 3, and wherein the one or moresubstitutions provides a variant having an IF of ≥1.2 for a measure ofstability and an IF of ≥1.0 for a measure of specific activity in ModelA and/or Model X detergent compositions.

In one embodiment, the variant comprises a substitution one or morepositions selected from the group consisting of; H1, D3, G7, N16, V17,Q22, H28, N33, N36, T40, W48, S52, Q53, N54, D55, V56, G59, A60, V75,R76, N95, V99, G109, A111, D112, F113, T114, R116, V117, Q118, V122,N123, P124, N126, N128, V131, Q136, I137, E138, A139, T141, G142, F143,N144, F145, P146, G149, Q151, S153, S154, K156, Y160, T165, D166, Q169,S170, R171, Q172, A174, R176, R181, K185, A186, T193, N195, Y203, D205,V206, D209, E212, V213, N215, N218, W220, G221, N226, F233, K242, D248,L250, G251, H252, G258, N260, K269, G273, E276, N277, T285, P292, L293,H294, N296, Q299, S304, G305, N306, R310, N311, L312, Q319, R320, H321,T327, T334, P336, E338, E341, G346, W347, L351, A354, Y363, G373, I374,P375, S376, D377, V379, S381, Y382, Q384, P388, L389, L390, Q394, Q395,Y398, R400, H402, Y404, V410, E416, N418, A419, S420, P422, M430, S431,G435, Q445, A447, M454, F473, and N475; wherein the positions correspondto amino acid positions in the amino acid sequence set forth in SEQ IDNO: 3, which one or more substitutions provides an improvement factor of≥1.5 for a measure of stability and optionally, an improvement factor of≥1.0 for a measure of specific activity in Model A and/or Model Xdetergent compositions.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; H1V, H1L, D3S, D3F, G7F, G7E,G7A, N16R, V17D, V17G, V17E, V17Q, V17A, V17T, V17L, Q22A, Q22Y, H28E,H28M, H28W, H28K, N33D, N36V, N36P, N36W, T40Q, T40S, W48C, W48I, W48H,W48A, S52M, S52V, S52C, S52F, S52A, S52D, S52K, S52N, S52E, S52T, Q53V,Q53L, Q53M, N54T, N54L, N54I, D55G, V56L, G59E, A60V, A60F, V75T, R76V,N95E, N95S, N95G, N95D, N95R, V99G, V99E, G109D, G109F, A111S, D112K,F113K, T114L, R116Q, R116K, V117G, V117I, V117M, Q118V, Q118G, Q118E,Q118F, Q118R, V122L, N123K, N123V, P124G, N126T, N128S, N128E, N128P,N128G, N128A, V131I, Q136E, Q136W, Q136G, I137V, E138T, E138L, A139S,A139G, A139V, A139T, A139P, T141S, T141G, G142R, G142A, F143L, N144G,N144R, F145A, F145N, F145Q, F145T, F145H, P146L, P146M, P146T, G149Y,G149L, G149A, Q151L, S153T, S154M, S154E, S154A, S154V, K156F, K156L,K156S, K156G, Y160H, T165I, D166S, Q169L, Q169S, Q169V, Q169F, S170A,S170V, S170Q, S170W, S170G, S170F, S170T, S170L, R171A, R171M, Q172L,Q172E, Q172C, Q172R, Q172G, Q172W, A174K, R176Y, R176M, R176T, R176K,K179L, R181A, R181G, R181D, K185G, A186S, A186D, A186T, A186Q, A186V,T193S, T193D, T193R, T193G, N195T, N195F, Y203W, Y203L, D205A, D205L,V206A, V206S, V206K, D209S, D209M, D209V, D209Q, D209L, D209I, E212G,E212I, E212C, E212K, V213W, V213R, V213F, N215C, N215T, N218D, N218R,N218L, N218M, W220L, W220I, W220P, W220E, W220R, W220A, W220S, G221A,N226C, N226M, N226E, N226K, N226R, F233T, F233P, F233M, K242A, D248G,D248R, L250Y, G251K, G251M, G251Q, H252F, H252V, G258D, N260W, N260R,K269W, G273E, E276L, N277D, T285M, T285P, P292L, P292G, L293G, L293Q,H294S, N296H, Q299L, Q299R, Q299P, S304G, S304L, S304F, S304A, S304M,G305F, G305I, N306V, N306L, N306D, N306S, N306T, R310T, N311C, N311Y,L312F, L312V, Q319H, Q319D, Q319F, R320V, H321R, H321S, T327V, T327E,T334S, P336L, P336W, P336S, P336K, E338A, E338H, E341M, E341F, E341K,E341S, G346S, W347L, W347R, W347V, W347C, L351H, L351Q, A354C, Y363R,Y363V, G373Q, G373W, G373R, I374Y, I374S, I374M, I374L, P375E, P375S,P375V, P375R, S376R, S376F, S376E, S376T, D377T, V379L, V379S, S381V,Y382R, Q384E, P388S, P388R, P388K, P388L, L389A, L389V, L389F, L389M,L390V, L390C, Q394N, Q394L, Q394T, Q394M, Q395R, Y398K, R400E, R400I,H402T, Y404F, Y404W, V410E, E416Q, N418M, N418G, N418R, N418F, A419M,A419L, A419Q, A419R, S420M, P422Y, P422H, P422D, P422R, M430L, S431A,S431F, G435S, Q445R, A447S, M454S, F473V, and N475G, wherein numberingis according to the amino acid sequence set forth in SEQ ID NO: 3, andwherein the one or more substitutions provides a variant having an IF of≥1.5 for a measure of stability and optionally, an IF of ≥1.0 for ameasure of specific activity in Model A and/or Model X detergentcompositions.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; H1V, G7F, G7E, Q22A, H28E, T40Q,W48C, W48I, S52M, S52V, Q53V, Q53L, V56L, G59E, V75T, N95E, N95S, N95G,R116Q, V117G, N128S, N128E, A139S, T141S, T141G, F143L, F145A, P146L,Q151L, K156F, K156L, K156S, K156G, T165I, D166S, Q169L, S170A, S170V,R171A, Q172L, Q172E, K179L, R181A, R181G, K185G, A186S, A186D, T193S,Y203W, Y203L, D209S, D209M, D209V, E212G, E212I, V213W, N215C, N226C,K242A, N260W, K269W, P292L, L293G, Q299L, Q299R, N306V, L312F, L312V,R320V, T327V, P336L, W347L, I374Y, S376R, V379L, S381V, L390V, Q395R,N418M, N418G, A419M, M430L, and S431A, wherein numbering is according tothe amino acid sequence set forth in SEQ ID NO: 3, and wherein the oneor more substitutions provides a variant having an IF of ≥1.5 for ameasure of stability and an IF of ≥1.0 for a measure of specificactivity in Model A and/or Model X detergent compositions.

In one embodiment, the variant comprises a substitution in one or morepositions selected from the group consisting of; H1, H2, T5, G7, I9,Q11, E14, W15, N19, Q22, W24, N25, R26, H28, N29, N30, A31, L34, K35,I39, T40, A41, I42, I44, A47, W48, T51, S52, Q53, V56, G59, T74, V75,189, R90, S91, L92, N95, I97, Q98, Y100, K108, F113, R116, V117, N126,N128, Q129, G133, A139, W140, T141, F143, F145, P146, Q151, K156, R158,H161, T165, D166, Q169, S170, R171, Q172, L173, A174, N175, K179, R181,G184, K185, A186, T193, Y203, D209, H210, E212, V213, N215, L217, N218,R219, V222, N226, T227, L228, K242, K245, L250, H252, V253, T257, G258,K259, N260, V264, K269, L279, K281, M286, P292, L293, Y295, L297, Q299,N306, L312, G315, T316, V318, R320, H321, S323, T327, V329, P336, G337,E341, S342, V344, Q345, G346, W347, P350, L351, Q361, Y363, Y368, I374,S376, V379, P380, S381, D387, L390, A392, Q395, H402, F405, T414, E416,N418, A419, M430, S431, G435, W439, K446, V450, G456, S459, N465, G476,and W482; wherein the positions correspond to amino acid positions inthe amino acid seqeuence set forth in SEQ ID NO: 3, which one or moresubstitutions provides an improvement factor of ≥1.0 for a measure ofstability and an improvement factor of ≥1.0 for a measure of specificactivity in Model A detergent composition.

In a particular embodiment, the variant comprises one or more of thesubstitutions selected from the group consisting of; H1V, H2T, T5L, G7F,G7V, G7E, G7E, G7D, I9M, I9L, Q11L, Q11V, E14F, W15G, W15S, N19G, N19S,Q22C, Q22A, W24F, N25V, R26L, H28E, N29G, N29A, N30S, A31S, L34Y, K35L,I39L, T40Q, T40A, A41Q, I42V, I44T, A47G, W48C, W48I, W48Y, W48F, T51A,S52M, S52V, Q53V, Q53L, V56L, G59E, T74V, T74L, V75T, I89A, R90L, S91A,S91G, L92C, N95E, N95S, N95G, I97V, Q98V, Q98L, Y100W, K108V, F113M,F113V, F113E, F113Y, R116Q, V117G, N126G, N126A, N128S, N128E, Q129E,G133A, G133D, E138A, A139S, W140H, T141S, T141C, T141G, F143M, F143L,F145A, P146L, Q151L, K156F, K156Q, K156L, K156S, K156G, R158S, H161C,T165I, D166S, Q169L, S170A, S170V, R171A, Q172L, Q172E, L173I, A174V,N175G, K179L, R181A, R181G, G184D, K185G, A186S, A186D, T193S, Y203W,Y203L, D209S, D209M, D209V, H210N, E212G, E212I, V213C, V213W, N215C,L217S, N218S, R219L, V222E, N226C, T227L, L228F, K242A, F245G, L250C,H252C, V253A, V253L, T257V, G258P, K259L, N260W, N260G, V264L, K269W,L279Q, K281A, M286L, P292L, L293T, L293G, Y295F, L297W, Q299S, Q299L,Q299R, N306V, L312F, L312V, G315V, T316S, V318L, R320V, R320C, H321T,S323N, T327V T327L, V329S, P336L, G337V, S342A, V344L, Q345L, G346R,W347L, P350S, P350Q, L351W, Q361V, Y363L, Y368H, Y372A, I374Y, S376R,S376L, V379L, P380V, S381V, D387Q, D387L, L390V, A392I, Q395E, Q395K,Q395R, H402L, F405G, T414A, E416V, N418M, N418G, A419M, M430L, S431A,G435N, W439E, W439R, W439N, K446G, V450T, G456Y, S459E, S459Q, N465L,N465R, D467C, G476Y, and W482F, wherein the numbering correspond toamino acid positions in the amino acid sequence set forth in SEQ ID NO:3, and wherein the substitutions provides a variant having an IF of ≥1.0for a measure of stability and an IF of ≥1.0 for a measure of specificactivity in Model A detergent composition.

In one embodiment, the variant comprises a substitution in one or morepositions selected from the group consisting of; H1, H2, T5, G7, I9,Q1I, N19, Q22, W24, N25, R26, H28, N29, N30, A31, L34, K35, I39, T40,A41, I42, I44, A47, W48, T51, S52, Q53, V56, G59, T74, V75, T81, I89,R90, S91, L92, N95, I97, Q98, Y100, K108, F113, R116, V117, N126, N128,Q129, G133, E138, A139, W140, T141, F143, F145, P146, Q151, K156, R158,H161, T165, D166, Q169, S170, R171, Q172, L173, A174, N175, K179, R181,G184, K185, A186, T193, E194, Y203, D209, H210, E212, V213, N215, L217,N218, R219, V222, N226, T227, L228, K242, K245, L250, H252, V253, T257,G258, K259, N260, V264, K269, L279, K281, M286, P292, L293, Y295, L297,Q299, N306, L312, G315, T316, V318, R320, H321, S323, T327, V329, P336,G337, E341, S342, V344, Q345, G346, W347, P350, Q361, Y363, I374, S376,V379, P380, S381, D387, L390, A392, Q395, H402, F405, E416, N418, A419,S431, G435, W439, K446, V450, G456, S459, N465, D467, G476, and W482,wherein the positions correspond to amino acid positions in the aminoacid sequence set forth in SEQ ID NO: 3, and wherein the one or moresubstitutions provide a variant having an IF of ≥1.0 for a measure ofspecific activity in Model X detergent composition and an IF of ≥1.0 fora measure of stability.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; H1V, H2T, T5L, G7F, G7E, G7V,G7E, G7D, I9M, I9L, Q11L, Q11V, N19G, N19S, Q22C, Q22A, W24F, N25V,R26L, H28E, N29G, N29A, N30S, A31S, L34Y, K35L, I39L, T40A, T40Q, A41Q,I42V, I44T, A47G, W48C, W48I, W48Y, W48F, T51A, S52V, S52M, Q53L, Q53V,V56L, G59E, T74L, T74V, V75T, T81G, I89A, R90L, S91A, S91G, L92C, N95G,N95E, N95S, I97V, Q98V, Q98L, Y100W, K108V, F113M, F113V, F113E, F113Y,R116Q, V117G, N126G, N126A, N128S, N128E, Q129E, G133A, G133D, E138A,A139S, W140H, T141C, T141G, T141S, F143M, F143L, F145A, P146L, Q151L,K156Q, K156F, K156L, K156S, K156G, R158S, H161C, T165I, D166S, Q169L,S170A, S170V, R171A, Q172E, Q172L, L173I, A174V, N175G, K179L, R181A,R181G, G184D, K185G, A186S, A186D, A186H, T193S, Y203L, Y203W, D209S,D209M, D209V, H210N, E212G, E212I, V213C, V213W, N215C, L217S, N218S,R219L, V222E, N226C, T227L, L228F, K242A, F245G, L250C, H252C, V253A,V253L, T257V, G258P, K259L, N260W, V264L, K269W, L279Q, K281A, M286L,P292L, L293T, L293G, Y295F, L297W, Q299S, Q299L, Q299R, N306V, L312F,L312V, G315V, T316S, V318L, R320C, R320V, H321T, S323N, T327L, T327V,V329S, P336L, G337V, S342A, V344L, Q345L, G346R, W347L, P350S, P350Q,Q361V, Y363L, I374Y, S376R, S376L, V379L, P380V, S381V, D387Q, D387L,L390V, A392I, Q395E, Q395K, Q395R, H402L, F405G, E416V, N418M, N418G,A419M, S431A, G435N, W439E, W439R, W439N, K446G, V450T, G456Y, S459Q,N465L, N465R, D467C, G476, and W482F wherein the positions correspond toamino acid positions in the amino acid sequence set forth in SEQ ID NO:3, wherein the one or more substitutions provides a variant having an IFof ≥1.0 for a measure of specific activity in Model X detergentcomposition and an IF of ≥1.0 for a measure of stability.

In one embodiment, the variant comprises a substitution in one or morepositions selected from the group consisting of; H1, G7, Q22, N25, R26,H28, N29, W48, S52, Q53, V56, G59, T74, V75, R90, N95, Q98, K108, F113,R116, V117, N128, Q129, G133, A139, W140, T141, F143, F145, P146, Q151,K156, T165, Q169, S170, R171, Q172, A174, K179, R181, K185, A186, T193,Y203, D209, E212, N215, L217, N226, K242, K259, N260, K269, K281, P292,L293, L297, N306, L312, G315, R320, H321, P336, V344, P350, Y363, Y368,S381, D387, A392, Q395, E416, N418, and S431; wherein the positionscorrespond to amino acid postions in the amino acid sequence set forthin SEQ ID NO: 3, which one or more substitutions provides an improvementfactor of ≥1.3 for a measure of stability and an improvement factor of≥1.3 for a measure of specific activity in Model A detergentcomposition.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; H1V, G7F, G7V, G7E, G7E, Q22A,N25V, R26L, H28E, N29G, W48C, W48I, W48F, S52M, S52V, Q53V, Q53L, V56L,G59E, T74L, V75T, R90L, N95E, N95S, N95G, Q98V, Q98L, K108V, F113V,R116Q, V117G, N128S, N128E, Q129E, G133A, G133D, A139S, W140H, T141S,T141C, T141G, F143M, F143L, F145A, P146L, Q151L, K156F, K156Q, K156L,K156S, K156G, T165I, D166S, Q169L, S170A, S170V, R171A, Q172L, Q172E,A174V, K179L, R181A, R181G, K185G, A186S, A186D, T193S, Y203W, Y203L,D209S, D209M, D209V, E212I, N215C, L217S, N226C, K242A, K259L, N260W,K269W, K281A, P292L, L293T, L293G, L297W, N306V, L312F, L312V, G315V,R320V, H321T, P336L, V344L, P350Q, Y363L, Y368H, S381V, D387L, A392I,Q395K, Q395R, E416V, N418M, N418G, and S431A, wherein numbering isaccording to the amino acid sequence set forth in SEQ ID NO: 3, andwherein the one or more substitutions provides a variant having an IF of≥1.3 for measure of stability and an IF of ≥1.3 for a measure ofspecific activity in Model A detergent composition.

In one embodiment, the variant comprises a substitution in one or morepositions selected from the group consisting of; H1, T5, G7, Q22, N25,R26, N29, W48, S52, Q53, T74, R90, N95, Q98, K108, F113, R116, V117,N128, Q129, G133, A139, W140, T141, F143, F145, P146, Q151, K156, T165,D166, Q169, S170, R171, Q172, A174, K179, R181, K185, A186, Y203, E212,N215, N226, K242, N260, K269, L279, K281, L293, Q299, N306, L312, G315,R320, V344, P350, Y363, I374, V379, S381, D387, A392, Q395, E416, N418,and S431, wherein the positions correspond to amino acid positions inthe amino acid sequence set forth in SEQ ID NO: 3, and wherein thesubstitution in the one or more positions provide a variant having an IFof ≥1.3 for a measure of specific activity in Model X detergentcomposition and an IF of ≥1.3 for a measure of stability.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; H1V, T5L, G7F, G7E, G7V, G7E,Q22A, N25V, R26L, N29G, W48C, W48I, S52V, S52M, Q53L, Q53V, T74L, R90L,N95G, N95E, N95S, Q98V, Q98L, K108V, F113V, R116Q, V117G, N128S, N128E,Q129E, G133A, G133D, A139S, W140H, T141C, T141G, T141S, F143M, F145A,P146L, Q151L, K156Q, K156F, K156L, K156S, K156G, T165I, D166S, Q169L,S170A, S170V, R171A, Q172E, Q172L, A174V, K179L, R181A, R181G, K185G,A186S, A186D, Y203L, Y203W, E212G, E212I, N215C, N226C, K242A, N260W,K269W, L279Q, K281A, L293T, L293G, Q299S, Q299L, N306V, L312F, L312V,G315V, R320V, V344L, P350Q, Y363L, I374Y, V379L, S381V, D387L, A392I,Q395K, Q395R, E416V, N418M, N418G, and S431A, wherein the positionscorrespond to amino acid positions in the amino acid sequence set forthin SEQ ID NO: 3, and wherein the one or more substitutions provide avariant having an IF of ≥1.3 for a measure of specific activity in ModelX detergent composition and an IF of ≥1.3 for a measure of stability.

In one embodiment, the variant comprises a substitution in one or morepositions selected from the group consisting of; H1, G7, Q22, S52, Q53,G59, R116, V117, N128, A139, T141, F143, F145, P146, Q151, K156, T165,D166, Q169, S170, R171, R181, K185, A186, Y203, E212, N215, K242, K269,N306, L312, and N418, wherein numbering is according to the amino acidsequence set forth in SEQ ID NO: 3, and wherein the substitution in oneor more positions provides a variant having an IF≥1.5 for a measure ofspecific activity in Model A detergent composition and an IF of ≥1.5 fora measure of stability.

In one embodiment, the variant comprises one or more substitutionselected from the group consisting of; H1V, G7E, Q22A, S52V, Q53V, Q53L,G59E, R116Q, V117G, N128S, N128E, A139S, T141S, T141G, F143L, F145A,P146L, Q151L, K156F, K156L, K156S, K156G, T165I, D166S, Q169L, S170A,S170V, R171A, R181A, R181G, K185G, A186S, A186D, Y203W, Y203L, E212I,N215C, K242A, K269W, N306V, L312F, L312V, and N418G, wherein numberingis according to the amino acid sequence set forth in SEQ ID NO: 3, andwherein the substitution in one or more positions provides a varianthaving an IF≥1.5 for a measure of specific activity in Model A detergentcomposition and an IF of ≥1.5 for a measure of stability.

In one aspect the present invention relates to an alpha-amylase variant,wherein the variant is a mature form of an alpha-amylase having amylaseactivity and comprising a substitution at one or more positions selectedfrom the group consisting of; E14, D20, Q22, G59, R116, A119, S132,G133, Y135, K156, R158, W159, Y160, K185, W187, Y202, D236, V238, K242,G337, E341, Y368, G369, D377, Y382, and K445; wherein the positionscorrespond to amino acids in the amino acid sequence set forth in SEQ IDNO: 3; and wherein the substitution provides at least one improvedproperty selected from the group consisting of improved washperformance, improved detergent stability, and improved thermostability.

The term “thermostability” as used herein, refers to the ability of anenzyme to retain activity after exposure to an elevated temperature. Thethermostability of an enzyme, such as an alpha-amylase or analpha-amylase variant, is measre by its half-life (tx) given in minutes,hours, or days, during which half the enzyme activity is lost underdefined conditions. The half-life may be calculated by measuringresidual alpha-amylase activity following exposure to (i.e. challengeby) an elevated temperature.

In another aspect, the present invention relates to an alpha-amylasevariant, wherein the variant is a mature form of an alpha-amylase havingamylase activity and comprising a substitution at one or more positionsselected from the group consisting of; E14, D20, Q22, G59, R116, A119,S132, G133, Y135, K156, R158, W159, Y160, K185, W187, Y202, D236, V238,K242, G337, E341, Y368, G369, D377, Y382, and K445, wherein thepositions correspond to amino acids in the amino acid sequence set forthin SEQ ID NO: 3; and wherein the substitution provides an alpha-amylasevariant having an improvement factor of ≥1.0 for a measure of specificactivity at pH 8.0 and 25° C., and an improvement factor of ≥1.0 for ameasure of stability in a detergent composition and for thermostability.

In one embodiment, the variant comprises one or more substitutionsselected from the group consisting of; D377V, W159D, K242E, R158Y,A119Y, D236P, R158E, Y135D, W187G, Y382L, R158V, G369I, G337M, K156G,W159E, Y160E, R158T, G59A, Y202S, K185V, S132D, Q22A, K156E, R116F,K185G, K242S, G133D, G369R, Y382E, D20R, Y368L, E341I, K445E, Y203V,V238W, and E14M, wherein the positions correspond to amino acidpositions in the amino acid sequence set forth in SEQ ID NO: 3.

In one embodiment, the amino acid substituted with in the particularposition is selected from the group consisting of A, C, D, E, F, G, H,I, K, L, M, N, P, Q, R, S, T, V, W, and Y, with the proviso that theamino acid substituted with is different from the naturally-occurringamino acid in said particular position.

The term “naturally-occurring amino acid” as used herein, refers to anyamino acid from the group of A, C, D, E; F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, and Y, which is found in the parent alpha-amylase beforeany modification, e.g. in the wild-type amino acid sequence of thealpha-amylase.

In certain embodiments, the variant may comprise a pairwise deletionwithin a particular loop of the alpha-amylase which has been found tofurther stabilize the alpha-amylase. Thus, in one embodiment, thevariant further comprises a pairwise deletion of the amino acid residuescorresponding to R181, G182, D183 and G184 of the amino acid sequenceset forth in SEQ ID NO: 3, with the proviso that when the amino acids inpositions corresponding to R181 and/or G184 has been substituted, thepairwise deletion is in the positions corresponding to G182 and D183 ofthe amino acid sequence set forth in SEQ ID NO: 3.

The term “pairwise deletion” as used herein, refers to one deletion intwo separate positions. Such positions may be adjacent to one anotherbut are not limited to such adjacent pairs. A pairwise deletion maythus, also be deletion of one amino acid and another amino acid whichmay be up to three amino acids further downstream or upstream from thefirst deletion. Accordingly, in one embodiment, the variant comprises apairwise deletion of the amino acids residues selected from the groupconsisting of; R181+G182, R181+D183, R181+G184, G182+D183, G182+G182,and D183+G184, wherein numbering is according to the amino acid sequenceset forth in SEQ ID NO: 3.

In a particular embodiment, the pairwise deletion is in the positionscorresponding to positions G182 and D183 of the amino acid sequence setforth in SEQ ID NO: 3.

In a further aspect, the present invention relates to an alpha-amylasevariant, wherein said variant is a mature form having amylase activityand comprising a) a deletion and/or substitution at two or morepositions corresponding to positions R181, G182, D183, and G184 of theamino acid sequence set forth in SEQ ID NO: 3; and b) a substitution atone or more positions selected from the group consisting of; H1, H2, D3,G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19,D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34,K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48,K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66,E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83,E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98,V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115,R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127,N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139,W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153,S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165,D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177,Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191,D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204,D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218,R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230,D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249,L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261,F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275,E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287,A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301,N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314,G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328,V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347,F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362,Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376,D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390,K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405,D406, H407, W408, D409, V410, I411, T414, R415, E416, N418, A419, S420,H421, P422, S424, G425, L426, A427, T428, I429, M430, S431, D432, G435,G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448,V450, W451, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464,N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482;wherein the positions correspond to amino acid positions in the aminoacid sequence set forth in SEQ ID NO: 3.

In one embodiment, the variant comprises a) a pairwise deletion of theamino acids residues selected from the group consisting of; R181+G182,R181+D183, R181+G184, G182+D183, G182+G182, and D183+G184, whereinnumbering is according to the amino acid sequence set forth in SEQ IDNO: 3, and b) one or more substitutions selected from the groupconsisting of; H1V, H1L, H1G, H1S, H1C, H2T, H2L, H2M, H2E, H2P, H2G,D3R, D3Y, D3W, D3S, D3F, D3C, D3A, G4Q, G4D, G4R, T5L, T5F, T5W, T5Q,T5G, T5E, T5V, G7F, G7V, G7E, G7E, G7D, G7L, G7A, G7R, G7S, G7N, T8A,T8S, I9C, I9M, I9L, M10L, Q11C, Q11L, Q11V, Q11M, Y12V, Y12A, Y12F,F13Y, F13I, E14Q, E14L, E14M, E14S, E14F, W15G, W15S, W15I, W15F, W15H,N16R, N16G, N16Q, V17D, V17G, V17E, V17Q, V17A, V17T, V17L, P18L, N19G,N19S, N19Y, N19T, N19V, D20R, Q22C, Q22A, Q22Y, Q22N, Q22S, Q22M, Q22T,Q22E, H23W, W24H, W24F, N25V, N25R, N25L, N25T, N25S, N25G, N25E, N25E,R26L, R26E, R26G, R26D, R26M, R26I, R26S, R26W, R26A, R26F, L27A, L27M,H28A, H28E, H28R, H28M, H28W, H28K, H28I, N29G, N29K, N29V, N29W, N29C,N29L, N29A, N30S, N30R, N30H, N30L, N30T, N30E, A31W, A31S, A31G, A31R,A31V, Q32D, Q32G, Q32L, Q32A, N33F, N33A, N33R, N33D, L34F, L34Y, K35L,K35E, K35W, K35Q, N36R, N36Q, N36A, N36E, N36V, N36P, N36W, N36T, A37R,A37G, A37T, A37S, A37E, G38N, G38M, G38Y, G38K, 139M, I39L, I39V, T40D,T40E, T40Q, T40A, T40S, A41E, A41N, A41M, A41Q, A41D, I42A, I42V, I42L,W43R, I44T, I44L, I44V, P45A, P46G, A47S, A47G, A47T, W48G, W48L, W48C,W48I, W48H, W48A, W48M, W48P, W48Q, W48Y, W48F, K49Q, G50P, G50S, G50N,T51S, T51G, T51P, T51L, T51C, T51A, S52M, S52V, S52C, S52F, S52A, S52D,S52K, S52N, S52E, S52T, S52I, S52L, Q53V, Q53L, Q53M, Q53G, N54G, N54K,N54V, N54T, N54L, N54I, N54S, D55G, D55V, D55I, V56L, V56M, V56T, G59E,G59S, G59A, A60V, A60F, A60S, A60W, A60L, A60G, A60T, Y61F, L63V, L63P,L63I, L63H, L63K, L63C, L63G, L63A, Y64W, Y64S, Y64V, Y64I, Y64H, Y64M,Y64C, Y64R, Y64G, Y64L, Y64A, L66M, E68D, E68S, E68M, E68C, E68T, E68V,E68Q, E68Y, F69H, F69W, N70G, N70A, N70T, N70S, N70E, Q71N, Q71E, Q71D,Q71I, Q71V, K72Y, K72R, G73V, G73C, G73L, G73E, T74V, T74L, T74N, V75T,V75E, V75G, R76S, R76G, R76A, R76V, T77A, T77S, T77V, K78L, K78T, K78A,T81W, T81L, T81G, K82A, K82G, A83G, A83E, A83Q, A83R, A83S, A83F, A83V,E84F, E84D, E84M, E84W, L85V, E86L, E86D, E86W, E86R, R87M, R87A, R87E,A88S, A88G, A88V, I89H, I89P, I89A, I89L, R90L, R90G, S91E, S91A, S91G,S91V, S91T, L92C, L92F, K93W, K93G, K93L, N95L, N95W, N95C, N95E, N95S,N95G, N95D, N95R, G96Q, G96R, 197M, I97V, I97L, Q98C, Q98M, Q98T, Q98V,Q98L, Q98F, Q98W, Q98P, Q98H, V99I, V99L, V99S, V99T, V99G, V99E, Y100V,Y100F, Y100W, V103G, V103A, V103S, V103T, H107Q, H107M, K108V, G109A,G109D, G109F, G109S, G110C, A111S, D112V, D112F, D112C, D112K, D112E,F113L, F113M, F113V, F113E, F113K, F113C, F113D, F113Q, F113Y, T114S,T114V, T114L, E115C, E115A, R116N, R116L, R116I, R116D, R116T, R116G,R116E, R116S, R116C, R116V, R116Q, R116K, R116F, R116H, R116W, V117C,V117L, V117S, V117W, V117A, V117G, V117I, V117M, V117E, Q118V, Q118G,Q118E, Q118F, Q118R, A119Y, A119W, V120I, V120D, V120L, V120C, V120A,V120N, E121P, E121G, E121V, E121M, E121D, V122E, V122P, V122L, V122I,V122F, N123G, N123A, N123E, N123W, N123T, N123P, N123K, N123V, P124R,P124G, Q125E, Q125T, N126E, N126G, N126A, N126R, N126T, R127G, R127S,R127C, R127W, R127L, R127V, R127F, R127Y, N128C, N128S, N128E, N128P,N128G, N128A, Q129L, Q129C, Q129E, E130C, V131L, V131E, V131I, S132A,S132H, S132P, S132C, S132E, S132D, A132Q, S132N, G133S, G133T, G133L,G133V, G133I, G133E, G133A, G133D, G133R, T134V, T134D, T134E, Y135L,Y135V, Y135S, Y135R, Y135T, Y135W, Y135K, Y135G, Y135P, Y135M, Y135D,Y135H, Y135F, Y135E, Q136E, Q136W, Q136G, I137A, I137S, I137G, I137V,E138A, E138Y, E138T, E138L, A139M, A139S, A139G, A139V, A139T, A139P,W140Y, W140S, W140A, W140H, T141M, T141S, T141C, T141G, G142D, G142E,G142R, G142A, F143M, F143L, F143I, N144C, N144M, N144K, N144G, N144R,F145C, F145S, F145A, F145N, F145Q, F145T, F145H, P146S, P146V, P146C,P146L, P146M, P146T, G147V, G147P, G147D, G147S, G147I, G147L, G147C,G149C, G149Y, G149L, G149A, N150C, N150D, N150E, Q151T, Q151C, Q151L,A151I, S153C, S153G, S153T, S154M, S154E, S154A, S154V, F155H, F155T,F155L, F155W, K156Y, K156V, K156A, K156D, K156W, K156P, K156E, K156C,K156F, K156Q, K156L, K156S, K156G, K156N, K156T, W157L, R158M, R158A,R158G, R158Q, R158W, R158L, R158T, R158V, R158E, R158Y, R158S, W159N,W159G, W159T, W159C, W159E, W159D, W159S, W159H, Y160Q, Y160M, Y160T,Y160A, Y160S, Y160V, Y160G, Y160I, Y160L, Y160E, Y160W, Y160R, Y160H,H161N, H161C, F162M, F162L, F162Y, F162I, D163S, D163K, D163A, D163R,D163L, G164S, G164T, G164C, T165L, T165M, T165S, T165G, T165I, T165V,T165E, D166T, D166N, D166S, W167Y, W167G, W167D, W167F, D168M, D168S,D168G, Q169A, Q169L, Q169S, Q169V, Q169F, Q169E, S170A, S170V, S170Q,S170W, S170G, S170F, S170T, S170L, R171F, R171S, R171K, R171V, R171A,R171M, Q172S, Q172L, Q172E, Q172C, Q172R, Q172G, Q172W, Q172K, L173G,L173N, L173M, L173S, L173Y, L173V, L173I, L173A, L173F, A174G, A174V,A174L, A174M, A174K, A174S, N175L, N175V, N175I, N175G, N175S, R176E,R176W, R176G, R176M, R176Y, R176M, R176T, R176K, I177L, I177S, I177G,Y178V, Y178R, Y178K, Y178W, K179C, K179E, K179S, K179W, K179A, K179G,K179T, K179F, K179L, F180A, F180L, R181K, R181P, R181S, R181V, R181Q,R181E, R181A, R181G, R181D, G184L, G184R, G184V, G184T, G184D, K185A,K185D, K185N, K185V, K185G, A186C, A186S, A186D, A186T, A186Q, A186V,A186N, A186E, A186H, W187G, D188Q, D188G, D188R, D188M, D188L, D188V,W189R, W189A, W189G, W189C, E190D, E190N, E190A, V191M, V191A, D192E,D192Q, D192S, T193K, T193V, T193C, T193P, T193S, T193D, T193R, T193G,E194S, E194D, E194L, E194Q, N195R, N195A, N195T, N195F, G196A, G196V,G196P, N197G, N197V, N197Y, N197C, N197D, Y198W, Y198A, Y198G, D199G,D199S, D199A, Y200W, Y200F, Y200C, Y200V, Y200T, Y200L, Y200A, Y200G,Y200S, M202T, M202L, Y203F, Y203G, Y203C, Y203I, Y203Q, Y203H, Y203T,Y203M, Y203S, Y203V, Y203W, Y203L, A204P, A204R, A204C, A204H, A204W,A204F, A204L, A204Y, A204D, A204G, D205C, D205R, D205A, D205L, V206H,V206Q, V206P, V206A, V206S, V206K, V206L, V206I, V206M, V206F, V206Y,M208R, M208P, M208L, M208C, D209C, D209E, D209H, D209F, D209Y, D209S,D209M, D209V, D209Q, D209L, D209I, H210F, H210V, H210A, H210W, H210L,H210G, H210D, H210E, H210Q, H210N, P211G, P211W, P211C, E212F, E212M,E212D, E212G, E212I, E212Y, E212R, E212C, E212K, V213M, V213T, V213G,V213C, V213W, V213R, V213F, I214P, I214V, N215D, N215V, N215C, N215T,L217V, L217C, L217P, L217T, L217G, L217S, L217A, L217W, N218S, N218D,N218R, N218L, N218M, R219H, R219W, R219S, R219I, R219A, R219M, R219T,R219E, R219Y, R219D, R219C, R219L, W220L, W220I, W220P, W220E, W220R,W220A, W220S, G221S, G221A, V222F, V222C, V222E, V222L, W223Y, W223F,W223L, Y224L, A225K, N226Q, N226C, N226M, N226E, N226K, N226R, T227S,T227M, T227C, T227L, T227Y, T227R, L228A, L228F, L228M, N229T, N229S,L230S, L230A, L230T, L230V, D231E, D231C, D231G, D231P, F233A, F233L,F233T, F233P, F233M, V238C, V238W, V238A, H240W, I241L, K242C, K242Y,K242P, K242W, K242G, K242L, K242R, K242S, K242E, K242A, K242H, S244Q,F245C, F245V, F245D, F245G, F245A, M246L, M246F, M246V, M246I, R247Y,R247G, R247M, D248G, D248R, W249L, W249F, L250W, L250T, L250A, L250C,L250F, L250Y, G251K, G251M, G251Q, H252E, H252W, H252C, H252T, H252F,H252V, V253S, V253W, V253A, V253L, R254Q, R254S, G255C, G255E, G255L,Q256P, T257V, G258V, G258C, G258I, G258L, G258P, G258F, G258D, K259V,K259Q, K259I, K259L, K259A, K259S, K259D, K259C, N260E, N260D, N260C,N260W, N260S, N260R, N260G, L261G, L261D, L261T, F262Q, F262L, F262E,F262R, F262P, F262V, A263L, A263C, V264T, V264L, Y267V, Y267L, W268V,W268Y, K269L, K269W, K269Q, K269R, N270D, N270A, N270M, N270G, D271E,D271V, D271C, L272M, G273W, G273R, G273V, G273E, A274S, A274D, A274E,L275G, L275E, L275A, L275R, L275K, E276R, E276L, N277D, Y278G, Y278S,L279V, L279G, L279Q, L279M, S280C, S280R, S280E, S280D, K281L, K281W,K281G, K281A, T282G, T282A, T282C, T282R, T282I, N283G, N283E, N283Q,N283V, N283A, W284T, W284I, W284S, W284E, W284C, W284V, W284A, T285E,T285F, T285S, T285W, T285M, T285P, M286D, M286L, M286C, S287V, S287Q,S287T, S287E, S287P, S287L, S287W, S287C, A288L, A288Y, A288V, V291T,P292A, P292Q, P292R, P292L, P292G, L293A, L293E, L293T, L293G, L293Y,L293Q, H294G, H294A, H294W, H294Q, H294S, Y295H, Y295S, Y295I, Y295A,Y295F, N296A, N296Y, N296M, N296F, N296S, N296K, N296H, L297S, L297W,L297H, Y298V, Y298P, Q299I, Q299S, Q299L, Q299R, Q299A, Q299H, Q299P,A300V, A300C, S301A, S301R, S301Q, N302I, N302T, N302M, N302S, S303I,S303E, S303A, S303M, S304I, S304P, S304G, S304L, S304F, S304A, S304M,G305V, G305L, G305S, G305Q, G305F, G305I, N306V, N306L, N306D, N306S,N306T, Y307F, Y307W, M309L, R310A, R310T, N311L, N311R, N311C, N311Y,L312F, L312V, L312I, L313S, L313M, L313I, N314Y, N314F, N314D, G315I,G315V, G315T, G315R, G315F, T316K, T316S, T316C, T316G, V318C, V318E,V318A, V318L, Q319L, Q319I, Q319A, Q319P, Q319H, Q319D, Q319F, R320L,R320V, R320C, R320A, R320S, H321E, H321V, H321C, H321T, H321G, H321R,H321S, P322E, P322C, S323N, A325V, A325S, V326I, V326G, V326A, V326C,V326T, T327S, T327V, T327L, T327Q, T327E, F328M, F328C, V329A, V329S,V329C, V329I, N331C, N331V, N331I, T334A, T334S, P336M, P336E, P336D,P336R, P336L, P336W, P336S, P336K, G337T, G337C, G337M, G337V, G337F,E338T, E338A, E338H, E341C, E341D, E341I, E341P, E341M, E341F, E341K,E341S, E341T, S342G, S342T, S342A, V344S, V344L, Q345K, Q345C, Q345V,Q345I, Q345L, Q345W, Q345N, G346H, G346A, G346L, G346N, G346Q, G346V,G346R, G346S, W347L, W347R, W347V, W347C, F348Q, F348S, F348C, F348P,F348M, K349G, K349A, K349Y, K349L, K349S, P350L, P350G, P350S, P350Q,P350D, P350E, L351M, L351S, L351W, L351H, L351Q, A352S, A352C, A354C,T355V, T355A, T355L, T355Y, T355F, L357C, L357T, L357S, L357H, T358V,T358F, T358S, T358L, E360S, E360D, E360C, E360G, E360A, E360M, Q361P,Q361R, Q361A, Q361E, Q361V, Q361G, Q361D, Q361S, G362Y, G362R, G362C,G362N, G362M, G362V, Y363Q, Y363I, Y363A, Y363L, Y363R, Y363V, Q365D,Q365M, Q365L, Q365T, Q365C, Q365S, V366L, V366A, V366I, V366C, F367L,F367S, F367A, Y368M, Y368T, Y368, Y368L, Y368A, Y368S, Y368Q, Y368V,Y368H, G369K, G369S, G369A, G369E, G369R, G369I, G369L, D370A, D370T,Y372S, Y372I, Y372L, Y372G, Y372A, G373C, G373S, G373T, G373Q, G373W,G373R, I374Y, I374S, I374M, I374L, P375L, P375A, P375T, P375E, P375S,P375V, P375R, S376A, S376N, S376P, S376W, S376R, S376L, S376V, S376I,S376Q, S376F, S376E, S376T, D377C, D377L, D377V, D377S, D377G, D377T,V379F, V379P, V379L, V379S, P380L, P380G, P380E, P380A, P380I, P380V,P380Y, S381N, S381L, S381A, S381I, S381V, Y382I, Y382A, Y382E, Y382V,Y382L, Y382K, Y382R, R383G, R383S, R383P, R383V, Q384A, Q384P, Q384L,Q384H, Q384R, Q384Y, Q384S, Q384V, Q384T, Q384G, Q384E, I386T, I386L,I386M, I386W, I386V, D387H, D387V, D387Q, D387L, P388G, P388T, P388M,P388W, P388V, P388S, P388R, P388K, P388L, L389S, L389H, L389A, L389V,L389F, L389M, L390S, L390F, L390I, L390G, L390V, L390T, L390N, L390H,L390C, K391L, K391Y, K391A, K391Q, A392I, A392M, A392T, Q394S, Q394R,Q394N, Q394L, Q394T, Q394M, Q395W, Q395N, Q395L, Q395E, Q395K, Q395R,Q395V, Q395S, Q395M, Y396T, Y396L, Y396R, Y396W, A397C, A397G, A397S,Y398S, Y398C, Y398N, Y398K, R400L, R400C, R400K, R400S, R400M, R400E,R400I, H402V, H402E, H402M, H402A, H402S, H402P, H402Y, H402L, H402D,H402R, H402T, D403E, D403P, D403Q, D403I, Y404K, Y404F, Y404W, F405C,F405V, F405I, F405P, F405W, F405G, F405A, D406E, D406V, D406T, D406M,D406S, H407C, H407D, W408R, W408P, W408C, W408Y, W408G, W408D, D409G,V410I, V410Q, V410L, V410E, I411A, I411L, I411V, T414V, 414A, R415F,R415L, R415A, R415Q, E416C, E416V, E416L, E416A, E416Q, N418L, N418A,N418C, N418M, N418G, N418R, N418F, A419E, A419C, A419M, A419G, A419V,A419L, A419Q, A419R, S420E, S420T, S420V, S420M, H421V, H421C, H421A,H421R, H421T, P422Y, P422H, P422D, P422R, S424C, S424A, S424L, G425A,G425V, L426S, L426A, A427V, A427S, T428L, T428N, T428A, T428G, I429V,I429A, M430R, M430L, M430A, M430T, S431A, S431T, S431F, D432Q, D432P,G435E, G435N, G435M, G435I, G435S, G436M, G436E, G436L, G436T, S437G,S437Q, S437L, K438T, W439C, W439H, W439E, W439A, W439R, W439T, W439N,W439T, W439R, W439N, M440L, Y441N, Y441H, Y441Q, Y441K, V442I, R444A,R444S, R444L, R444V, R444P, Q445L, Q445S, Q445C, Q445E, Q445R, K446Q,K446G, K446T, K446P, A447G, A447Q, A447E, A447T, A447R, A447S, G448S,G448R, V450A, V450T, V450Q, V450R, W451L, H452A, H452T, H452C, H452G,H452Y, H452R, H452V, D453E, M454R, M454L, M454V, M454Q, M454S, T455L,T455S, G456V, G456R, G456Y, G456K, G456L, N457V, N457I, N457G, N457R,S459V, S459C, S459P, S459M, S459T, S459E, S459Q, S459R, G460S, G460D,G460E, T463C, T463V, T463L, T463D, T463H, T463M, T463A, T463Q, I464V,N465G, N465Q, N465L, N465R, Q466L, Q466C, Q466E, D467V, D467C, W469M,W469E, W469T, W469N, H471Q, F473Y, F473C, F473R, F473E, F473G, F473T,F473V, N475T, N475H, N475G, G476F, G476K, G476Y, G477Q, G477F, G477K,G477L, V481Q, V481S, and W482F, wherein the numbering is according tothe amino acid sequence set forth in SEQ ID NO: 3.

The variants according to the present invention may further comprise oneor more additional alterations than those described above at one or more(e.g., several) other positions.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for alpha-amylase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

In one embodiment, the variant comprises a modification in 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, I2, 13, 14, 15, 16, 17, 18, 19, or 20 positions.

In one embodiment, the variant comprises a substitution at 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 positions.

In any embodiment disclosed herein, the stability may be determined asdescribed in Example [stability], and the specific activity may bedetermined as described in Example [spec. act.] when using Model Adetergent composition or Model X detergent composition.

The variants according to the present invention may consist of 400 to485 amino acids, e.g., 410 to 485, 425 to 485, and 440 to 485 aminoacids.

Polynucleotides

The present invention also relates to polynucleotides encoding a variantof the present invention. Thus, in particular, the present inventionrelates to a polynucleotide encoding a variant comprising an alterationin one or more positions corresponding to the positions H1, H2, D3, G4,T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20,Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35,N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49,G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68,F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84,L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99,Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116,V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128,Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140,T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154,F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166,W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178,K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192,T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205,V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219,W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231,F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250,G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262,A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276,N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288,V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302,S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315,T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329,N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348,K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363,Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377,V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391,A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406,H407, W408, D409, V410, I411, T414, R415, E416, N418, A419, S420, H421,P422, S424, G425, L426, A427, T428, I429, M430, S431, D432, G435, G436,S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448, V450,W451, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465,Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482, whereinthe positions correspond to amino acid positions in the amino acidsequence set forth in SEQ ID NO: 3.

The term “polynucleotides encoding” as used herein, refers to apolynucleotide that encodes a mature polypeptide having alpha-amylasehaving alpha-amylase activity. In one aspect, the polypeptide codingsequence is the nucleotide sequence set forth in SEQ ID NO: 1.

In one embodiment, the polynucleotide encoding a variant according tothe present invention as at least 80%, such as at least 85%, such as atleast 90%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, such as at least 99% but less than 100%sequence identity to the polynucleotide of SEQ ID NO: 1.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences. Thus, in particular, the present inventionrelates to a nucleic acid construct comprising a polypeptide encoding avariant comprising an alteration in one or more positions correspondingto the positions H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13,E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28,N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42,W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56,G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75,R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91,L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110,A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122,N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134,Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146,G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160,H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172,L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186,W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198,D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212,V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225,N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244,F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256,T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270,D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282,N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296,L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309,R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322,S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341,S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355,L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370,Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384,I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398,R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414,R415, E416, N418, A419, S420, H421, P422, S424, G425, L426, A427, T428,I429, M430, S431, D432, G435, G436, S437, K438, W439, M440, Y441, V442,R444, Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456,N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475,G476, G477, V481, and W482 of the amino acid sequence set forth in SEQID NO: 3, wherein the polypeptide is operatively linked to one or morecontrol sequences.

The term “nucleic acid construct” as used herein, refers to a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or is modified to contain segments of nucleicacids in a manner that would not otherwise exist in nature or which issynthetic, which comprises one or more control sequences. A nucleic acidencompasse DNA, RNA, heteroduplexes, and sunthetic molecules capanble ofencoding a polypeptide. Nucleic acids may be single stranded or doublestranded, and may be chemical modifcations. The terms “nucleic acids”and “polynucleotide” may be used interchangeably, but constitute thesame meaning and purpose. Because the genetic code is degenerate, morethan one codon may be used to encode a particular amino acid, and thepresent compositions and methods encompass nucleotide sewuences whichencode a particular amino acid sequence. Unless otherwise indicated bycontext, nucleic acids are written left to right in 5′ to 3′orientation; amino acids sequences are written left to right in amino tocarboxy orientation, respectively.

The term “operably linked” as used herein, refers to a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter comprises transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

The term “promoter” as used herein, refers to a regulartory sequencethat is involved in binding RNA polymerase to initiate transcription ofa gene, and may be an inducible promoter or a constitutive promoter. Theskilled person would know of possible promoters that are suitable forthe particular nucleic acid construct used.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thus,the present invention relates to an expression vector, optionallyrecombinant, comprising a polynucleotide encoding a variant comprisingan alteration in one or more positions corresponding to the positionsH1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17,P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32,N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46,A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63,Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81,K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96,I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113,T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125,N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137,E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150,Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163,G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175,R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189,E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202,Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215,L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228,N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247,D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259,N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273,A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285,M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299,A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312,L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326,T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345,G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360,Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374,P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388,L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403,Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E416, N418,A419, S420, H421, P422, S424, G425, L426, A427, T428, I429, M430, S431,D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446,A447, G448, V450, W451, H452, D453, M454, T455, G456, N457, S459, G460,T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481,and W482 of the amino acid sequence set forth in SEQ ID NO: 3, apromotor, and transcriptional and translational stop signals.

The term “expression vector” as used herein, refers to a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

The various nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

The skilled person would know which expression vector is the mostsuitable for specific expression systems. Thus, the present invention isnot limited to any specific expression vector, but any expression vectorcomprising the polynucleotide encoding a variant according to theinvention is considered part of the present invention.

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. Thus, the present invention relates toa host cell, optionally a recombinant host cell, comprising apolynucleotide encoding a variant comprising an alteration in one ormore positions corresponding to the positions H1, H2, D3, G4, T5, G7,T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23,W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37,G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51,S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70,Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86,R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103,H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118,A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130,V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142,F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156,W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168,Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180,R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194,N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208,D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221,V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238,H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252,V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264,Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278,L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292,L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304,G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318,Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334,P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350,L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366,F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380,S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394,Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408,D409, V410, I411, T414, R415, E416, N418, A419, S420, H421, P422, S424,G425, L426, A427, T428, I429, M430, S431, D432, G435, G436, S437, K438,W439, M440, Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452,D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467,W469, H471, F473, N475, G476, G477, V481, and W482 of the amino acidsequence set forth in SEQ ID NO: 3, operably linked to one or morecontrol sequences that direct the production of the variant.

The term “host cell” as used herein, refers to any cell type that issusceptible to transformation, transfection, transduction, or the likewith a nucleic acid construct or expression vector comprising apolynucleotide of the present invention. The term “host cell”encompasses any progeny of a parent host cell that is not identical tothe parent host cell due to mutations that occur during replication.

A construct or vector comprising a polynucleotide is introduced into ahost cell so that the construct or vector is maintained as a chromosomalintegrant or as a self-replicating extrachromosomal vector as describedearlier. The choice of a host cell will to a large extent depend uponthe gene encoding the variant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

Methods According to the Invention

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant. Thus, the present invention relates to a method ofproducing a variant comprising an alteration in one or more positionscorresponding to the positions H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11,Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26,L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40,A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54,D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73,T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89,R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108,G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120,E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132,G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144,F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158,W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170,R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184,K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196,N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210,P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223,Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241,K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254,G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268,K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280,K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294,Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306,Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320,H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337,E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352,A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368,G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382,R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396,A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410,I411, T414, R415, E416, N418, A419, S420, H421, P422, S424, G425, L426,A427, T428, I429, M430, S431, D432, G435, G436, S437, K438, W439, M440,Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452, D453, M454,T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471,F473, N475, G476, G477, V481, and W482 of the amino acid sequence setforth in SEQ ID NO:3, wherein the method comprises the steps of a)cultivating a host cell according to the invention under conditionssuitable for expression of the variant, and b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the polypeptides having alpha amylase activity. Thesedetection methods include, but are not limited to, use of specificantibodies, formation of an enzyme product, or disappearance of anenzyme substrate. For example, an enzyme assay may be used to determinethe activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

In a particular embodiment, the method of producing a variant comprisesthe steps of a) introducing a deletion and/or substitution at two ormore positions corresponding to positions 181, 182, 183, and 184 of theamino acid sequence set forth in SEQ ID NO: 3; and b) introducing asubstitution at one or more positions selected from the group consistingof; H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16,V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31,Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45,P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61,L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78,T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95,G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112,F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124,Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136,I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149,N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162,D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174,N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188,W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200,M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214,N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227,L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246,R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258,K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272,G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284,T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298,Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311,L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325,V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344,Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358,E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373,I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387,P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402,D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E416,N418, A419, S420, H421, P422, S424, G425, L426, A427, T428, I429, M430,S431, D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445,K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457, S459,G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477,V481, and W482; wherein the positions correspond to amino acid positionsin the amino acid sequence set forth in SEQ ID NO: 3.

Furthermore, the present invention relates to methods for obtaining avariant, comprising introducing into a parent alpha-amylase having atleast 80% sequence identity to the polypeptide of SEQ ID NO: 4

(a) a substitution and/or deletion of two or more positions in theparent alpha-amylase said positions corresponding to positions 181, 182,183, and 184 of the mature polypeptide of SEQ ID NO: 3; and

(b) a substitution at one or more positions selected from the groupconsisting of; H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14,W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29,N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43,I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59,A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76,T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92,K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111,D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123,P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135,Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147,G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161,F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173,A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187,D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199,Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213,I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226,T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245,M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257,G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271,L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283,W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297,Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310,N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323,A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342,V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357,T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372,G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386,D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400,H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415,E416, N418, A419, S420, H421, P422, S424, G425, L426, A427, T428, I429,M430, S431, D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444,Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457,S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476,G477, V481, and W482; wherein the positions correspond to amino acidpositions in the amino acid sequence set forth in SEQ ID NO: 3,

wherein the resulting variant has at least 80%, such as at least 85%,such as at least 90%, such as at least 95%, such as at least 97%, butless than 100% sequence identity with the mature polypeptide of SEQ IDNOs: 3 or 4, wherein said variant has alpha-amylase activity;

(c) recovering said variant.

The variants may be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent polypeptideand subsequent ligation of an oligonucleotide containing the mutation inthe polynucleotide. Usually the restriction enzyme that digests theplasmid and the oligonucleotide is the same, permitting sticky ends ofthe plasmid and the insert to ligate to one another. See, e.g., Schererand Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure may be used in the presentinvention. There are many commercial kits available that can be used toprepare variants. The skilled person in the art is well-aware of suchcommercial kits and how to use them.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis may be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions may be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that may beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods may be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides may be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

The present invention also relates to a method of improving performance,such as wash performance, of a parent alpha-amylase having the aminoacid sequence of SEQ ID NO: 3 or 4, or having at least 65% sequenceidentity hereto, said method comprising the steps of a) introducing apairwise deletion and/or substitution at two or more positionscorresponding to positions 181, 182, 183, and 184 of the amino acidsequence set forth in SEQ ID NO: 3; and b) introducing a substitution atone or more positions selected from the group consisting of; H1, H2, D3,G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19,D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34,K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48,K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66,E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83,E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98,V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115,R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127,N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139,W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153,S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165,D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177,Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191,D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204,D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218,R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230,D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249,L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261,F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275,E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287,A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301,N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314,G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328,V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347,F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362,Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376,D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390,K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405,D406, H407, W408, D409, V410, I411, T414, R415, E416, N418, A419, S420,H421, P422, S424, G425, L426, A427, T428, I429, M430, S431, D432, G435,G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448,V450, W451, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464,N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482;wherein the positions correspond to amino acid positions in the aminoacid sequence set forth in SEQ ID NO: 3; said method thereby providingan alpha-amylase variant of said parent alpha-amylse, wherein saidvariant has at least 65%, such as at least 70%, such as at least 75%,such as at least 80%, such as at least 85%, such as at least 90%, suchas at least 95%, such as at least 97%, such as at least 99%, but lessthan 100%, sequence identity to the amino acid sequence as set forth inSEQ ID NOs: 3 or 4, and wherein said variant has alpha-amylase activityand improved performance as compared to said parent alpha-amylase,

wherein the variant has at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, at least 99%, but less than 100%sequence identity with the amino acid sequence set forth in SEQ ID NOs:3 or 4, and wherein the variant has alpha-amylase activity and improvedperformance compared to the parent alpha-amylase.

In one embodiment, the variant has at least 50%, such as at least 60%,or at least 70%, or at least 80%, or at least 90%, or at least 100% ofthe activity of the parent polypeptide having the amino acid sequence ofSEQ ID NO: 3.

In one embodiment, the activity is determined according to a Phadebasassay.

The alpha-amylase activity may be determined by a method using thePhadebas substrate (from for example Magle Life Sciences, Lund, Sweden).A Phadebas tablet includes interlinked starch polymers that are in theform of globular microspheres that are insoluble in water. A blue dye iscovalently bound to these microspheres. The interlinked starch polymersin the microsphere are degraded at a speed that is proportional to thealpha-amylase activity. When the alpha-amylase degrades the starchpolymers, the released blue dye is water soluble and concentration ofdye can be determined by measuring absorbance at 620 nm. Theconcentration of blue is proportional to the alpha-amylase activity inthe sample.

The variant sample to be analyzed is diluted in activity buffer with thedesired pH. Two substrate tablets are suspended in 5 mL activity bufferand mixed on magnetic stirrer. During mixing of substrate transfer 150μl to microtiter plate (MTP) or PCR-MTP. Add 30 μl diluted amylasesample to 150 μl substrate and mix. Incubate for 15 minutes at 37° C.The reaction is stopped by adding 30 μl 1M NaOH and mix. Centrifuge MTPfor 5 minutes at 4000×g. Transfer 100 μl to new MTP and measureabsorbance at 620 nm.

The alpha-amylase sample should be diluted so that the absorbance at 620nm is between 0 and 2.2, and is within the linear range of the activityassay.

Thus, in one embodiment, the activity is determined by by a methodcomprising the steps of;

-   -   a) incubating an alpha-amylase variant according to the        invention with a dyed amylose substrate for 15 minute at 37° C.;        and    -   b) measuring the absorption at OD 620 nm.

In a further embodiment, the activity is determined by a methodcomprising the steps of;

-   -   a) incubating an alpha-amylase variant according to the        invention with a dyed amylose substrate for 15 minute at 37° C.;        and    -   b) centrifuging the sample;    -   c) transferring the supernatant to reader plate, and measuring        the absorption at OD 620 nm.

In another aspect, the present invention relates to a method ofimproving the stability, such as stability in a detergent compositionand thermostability, of a parent alpha-amylase having the amino acidsequence of SEQ ID NO: 3 or 4, or having at least 65% sequence identityhereto, said method comprising the steps of a) introducing a pairwisedeletion and/or substitution at two or more positions corresponding topositions 181, 182, 183, and 184 of the amino acid sequence set forth inSEQ ID NO: 3; and b) introducing a substitution at one or more positionsselected from the group consisting of; H1, H2, D3, G4, T5, G7, T8, I9,M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24,N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38,I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52,Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71,K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87,A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107,K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119,V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131,S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143,N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157,R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169,S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181,G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195,G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209,H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222,W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240,I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253,R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267,W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279,S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293,H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305,N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319,R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336,G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351,A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367,Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381,Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395,Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409,V410, I411, T414, R415, E416, N418, A419, S420, H421, P422, S424, G425,L426, A427, T428, I429, M430, S431, D432, G435, G436, S437, K438, W439,M440, Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452, D453,M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469,H471, F473, N475, G476, G477, V481, and W482; wherein the positionscorrespond to amino acid positions in the amino acid sequence set forthin SEQ ID NO: 3; said method thereby providing an alpha-amylase variantof said parent alpha-amylse, wherein said variant has at least 80%, suchas at least 85%, such as at least 90%, such as at least 95%, such as atleast 97%, such as at least 99%, but less than 100%, sequence identityto the amino acid sequence as set forth in SEQ ID NOs: 3 or 4, andwherein said variant has alpha-amylase activity and improved stabilty ascompared to said parent alpha-amylase.

In one embodiment, the stability is determined according to a Phadebasassay.

In particular, the stability may be determined according to an assaycomprising the steps of;

-   -   a) incubating the variant to be tested at 43° C. for 16 hrs in a        detergent composition, such as Model A detergent composition,        and another sample at 4° C. for 16 hrs,    -   b) adding the sample comprising the variant to be tested to a        substrate plate,    -   c) measuring the absorbance at 620 nm, and    -   d) calculating the % residual activity as the ratio between the        sample incubated at 43° C. and the sample incubated at 4° C.

The stability may be determined by other methods known in the art.

In one embodiment, the variant has at least 50%, such as at least 60%,or at least 70%, or at least 80%, or at least 90%, or at least 100% ofthe activity of the parent alpha-amylase having the amino acid sequenceof SEQ ID NOs: 3 or 4.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.Thus, in one embodiment, the fermentation broth formulation or the cellcomposition comprises a polynucleotide encoding a variant comprising analteration in one or more positions corresponding to the positions H1,H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18,N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33,L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47,W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64,L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82,A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97,Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114,E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126,R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138,A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151,S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164,T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176,I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190,V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203,A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217,N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229,L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248,W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260,L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274,L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286,S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300,S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313,N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327,F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346,W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361,G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375,S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389,L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404,F405, D406, H407, W408, D409, V410, I411, T414, R415, E416, N418, A419,S420, H421, P422, S424, G425, L426, A427, T428, I429, M430, S431, D432,G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447,G448, V450, W451, H452, D453, M454, T455, G456, N457, S459, G460, T463,I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, andW482; wherein the positions correspond to amino acid positions in theamino acid sequence set forth in SEQ ID NO: 3, a nucleic acid constructencoding a variant comprising an alteration in one or more positionscorresponding to the positions H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11,Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26,L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40,A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54,D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73,T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89,R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108,G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120,E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132,G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144,F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158,W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170,R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184,K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196,N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210,P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223,Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241,K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254,G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268,K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280,K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294,Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306,Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320,H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337,E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352,A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368,G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382,R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396,A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410,I411, T414, R415, E416, N418, A419, S420, H421, P422, S424, G425, L426,A427, T428, I429, M430, S431, D432, G435, G436, S437, K438, W439, M440,Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452, D453, M454,T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471,F473, N475, G476, G477, V481, and W482; wherein the positions correspondto amino acid positions in the amino acid sequence set forth in SEQ IDNO: 3, or an expression vector encoding a variant comprising analteration in one or more positions corresponding to the positions H1,H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18,N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33,L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47,W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64,L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82,A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97,Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114,E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126,R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138,A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151,S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164,T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176,I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190,V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203,A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217,N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229,L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248,W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260,L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274,L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286,S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300,S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313,N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327,F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346,W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361,G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375,S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389,L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404,F405, D406, H407, W408, D409, V410, I411, T414, R415, E416, N418, A419,S420, H421, P422, S424, G425, L426, A427, T428, I429, M430, S431, D432,G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447,G448, V450, W451, H452, D453, M454, T455, G456, N457, S459, G460, T463,I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, andW482; wherein the positions correspond to amino acid positions in theamino acid sequence set forth in SEQ ID NO: 3. The fermentation brothproduct may further comprise additional ingredients used in thefermentation process, such as, for example, cells (including, the hostcells containing the gene encoding the polypeptide of the presentinvention which are used to produce the polypeptide of interest), celldebris, biomass, fermentation media and/or fermentation products. Insome embodiments, the composition is a cell-killed whole brothcontaining organic acid(s), killed cells and/or cell debris, and culturemedium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In one embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a particular embodiment, the first organicacid component is acetic acid, formic acid, propionic acid, a saltthereof, or a mixture of two or more of the foregoing and the secondorganic acid component is benzoic acid, cyclohexanecarboxylic acid,4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture oftwo or more of the foregoing.

In one embodiment, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may comprise theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition comprises the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis. In some embodiments, the cell-killed whole broth orcomposition comprises the spent cell culture medium, extracellularenzymes, and killed filamentous fungal cells. In some embodiments, themicrobial cells present in the cell-killed whole broth or compositionmay be permeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may comprise insoluble components, such as killed cells,cell debris, culture media components, and/or insoluble enzyme(s). Insome embodiments, insoluble components may be removed to provide aclarified liquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Enzyme Compositions

The present invention also relates to compositions comprising a variantaccording to the invention. Thus, the invention relates to a compositioncomprising a variant comprising an alteration in one or more positionscorresponding to the positions H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11,Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26,L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40,A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54,D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73,T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89,R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108,G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120,E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132,G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144,F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158,W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170,R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184,K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196,N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210,P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223,Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241,K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254,G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268,K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280,K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294,Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306,Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320,H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337,E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352,A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368,G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382,R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396,A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410,I411, T414, R415, E416, N418, A419, S420, H421, P422, S424, G425, L426,A427, T428, I429, M430, S431, D432, G435, G436, S437, K438, W439, M440,Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452, D453, M454,T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471,F473, N475, G476, G477, V481, and W482; wherein the positions correspondto amino acid positions in the amino acid sequence set forth in SEQ IDNO: 3.

In one embodiment, the composition comprises a variant comprising

a) a deletion and/or a substitution at two or more positionscorresponding to positions R181, G182, D183, and G184 of the amino acidsequence as set forth in SEQ ID NOs: 7 or 10, and

b) a substitution at one or more positions selected from the groupconsisting of; H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14,W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29,N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43,I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59,A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76,T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92,K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111,D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123,P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135,Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147,G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161,F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173,A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187,D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199,Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213,I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226,T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245,M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257,G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271,L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283,W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297,Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310,N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323,A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342,V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357,T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372,G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386,D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400,H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415,E416, N418, A419, S420, H421, P422, S424, G425, L426, A427, T428, I429,M430, S431, D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444,Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457,S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476,G477, V481, and W482; wherein the positions correspond to amino acidpositions in the amino acid sequence set forth in SEQ ID NO: 3.Preferably, the compositions are enriched in such a variant. The term“enriched” as used herein, refers to that the alpha-amylase activity ofthe composition has been increased, e.g., with an enrichment factor ofat least 1.1.

The compositions may comprise a variant of the present invention as themajor enzymatic component, e.g., a mono-component composition. Inanother embodiment, the composition further comprises at least onefurther active component.

The term “active component” as used herein, refers to any biological ornon-biological molecule which in itself is active. For example, anactive component is an enzyme.

Thus, in one embodiment, the further active component is an enzyme, suchas a protease, lipase, cellulose, pectate lyase and mannanase. Thus, thecompositions may comprise multiple enzymatic activities, such as one ormore (e.g., several) enzymes selected from the group consisting ofhydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase,e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase,amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase,carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase,chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase,laccase, lipase, lichinase, mannosidase, mutanase, oxidase, pectinolyticenzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, or xylanase.

In one embodiment, the composition is a detergent composition, such as aliquid or powder detergent composition.

In one embodiment, the composition is a liquid laundry or liquid dishwash composition, such as an Automatic Dish Wash (ADW) liquid detergentcomposition, or a powder laundry, such as a soap bar, or powder dishwash composition, such as an ADW unit dose detergent composition.

The choice of additional components is within the skills of the skilledperson in the art and includes conventional ingredients, including theexemplary non-limiting components set forth below. The choice ofcomponents may include, for fabric care, the consideration of the typeof fabric to be cleaned, the type and/or degree of soiling, thetemperature at which cleaning is to take place, and the formulation ofthe detergent product. Although components mentioned below arecategorized by general header according to a particular functionality,this is not to be construed as a limitation, as a component may compriseadditional functionalities as will be appreciated by the skilledartisan.

In one embodiment of the present invention, the variant of the presentinvention may be added to a detergent composition in an amountcorresponding to 0.001-100 mg of protein, such as 0.01-100 mg ofprotein, preferably 0.005-50 mg of protein, more preferably 0.01-25 mgof protein, even more preferably 0.05-10 mg of protein, most preferably0.05-5 mg of protein, and even most preferably 0.01-1 mg of protein perliter of wash liquor. The term “protein” in this context is contemplatedto be understood to include a variant according to the presentinvention.

A composition for use in automatic dish wash (ADW), for example, mayinclude 0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as0.05-5% of enzyme protein by weight of the composition.

A composition for use in laundry granulation, for example, may include0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as 0.05%-5% ofenzyme protein by weight of the composition.

A composition for use in laundry liquid, for example, may include0.0001%-10%, such as 0.001-7%, such as 0.1%-5% of enzyme protein byweight of the composition.

The variants of the invention as well as the further active components,such as additional enzymes, may be stabilized using conventionalstabilizing agents, e.g., a polyol such as propylene glycol or glycerol,a sugar or sugar alcohol, lactic acid, boric acid, or a boric acidderivative, e.g., an aromatic borate ester, or a phenyl boronic acidderivative such as 4-formylphenyl boronic acid, and the composition maybe formulated as described in, for example, WO92/19709 and WO92/19708.

In certain markets different wash conditions and, as such, differenttypes of detergents are used. This is disclosed in e.g. EP 1 025 240.For example, In Asia (Japan) a low detergent concentration system isused, while the United States uses a medium detergent concentrationsystem, and Europe uses a high detergent concentration system.

A low detergent concentration system includes detergents where less thanabout 800 ppm of detergent components are present in the wash water.Japanese detergents are typically considered low detergent concentrationsystem as they have approximately 667 ppm of detergent componentspresent in the wash water.

A medium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of detergent components are present in thewash water. North American detergents are generally considered to bemedium detergent concentration systems as they have approximately 975ppm of detergent components present in the wash water.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 4500-5000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range from1500 ppm to 6000 ppm of detergent components in the wash water. Suchdetergent compositions are all embodiments of the invention.

A variant of the present invention may also be incorporated in thedetergent formulations disclosed in WO97/07202, which is herebyincorporated by reference.

Examples are given herein of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

In particular, a composition according to the present invention furthercomprises a chelator.

The term “chelator” as used herein, refers to chemicals which formmolecules with certain metal ions, inactivating the ions so that theycannot react with other elements. Thus, a chelator may be defined as abinding agent that suppresses chemical activity by forming chelates.Chelation is the formation or presence of two or more separate bindingsbetween a ligand and a single central atom. The ligand may be anyorganic compound, a silicate or a phosphate. In the present context theterm “chelating agents” comprises chelants, chelating agent, chelatingagents, complexing agents, or sequestering agents that formswater-soluble complexes with metal ions such as calcium and magnesium.The chelate effect describes the enhanced affinity of chelating ligandsfor a metal ion compared to the affinity of a collection of similarnonchelating ligands for the same metal. Chelating agents having bindingcapacity with metal ions, in particular calcium (Ca2+) ions, and hasbeen used widely in detergents and compositions in general for wash,such as laundry or dish wash. Chelating agents have however shownthemselves to inhibit enzymatic activity. The term chelating agent isused in the present application interchangeably with “complexing agent”or “chelating agent” or “chelant”.

Since most alpha-amylases are calcium sensitive the presence ofchelating agents these may impair the enzyme activity. The calciumsensitivity of alpha-amylases can be determined by incubating a givenalpha-amylase in the presence of a strong chelating agent and analyzethe impact of this incubation on the activity of the alpha-amylase inquestion. A calcium sensitive alpha-amylase will lose a major part orall of its activity during the incubation. Chelating agent may bepresent in the composition in an amount from 0.0001 wt % to 20 wt %,preferably from 0.01 to 10 wt %, more preferably from 0.1 to 5 wt %.

Non-limiting examples of chelating agents are; EDTA, DTMPA, HEDP, andcitrate. Thus, in one embodiment, the composition comprises a variantaccording to the invention and a chelating agent, such as EDTA, DTMPA,HEDP or citrate.

The term “EDTA” as used herein, refers to ethylene-diamine-tetra-aceticacid which falls under the definition of “strong chelating agents”.

The term “DTMPA” as used herein, refers to diethylenetriaminepenta(methylene phosphonic acid). DTMPA can inhibit the scale formationof carbonate, sulfate and phosphate.

The term “HEDP” as used herein, refers to hydroxy-ethane diphosphonicacid, which falls under the definition of “strong chelating agents”.

The chelate effect or the chelating effect describes the enhancedaffinity of chelating ligands for a metal ion compared to the affinityof a collection of similar nonchelating ligands for the same metal.However, the strength of this chelate effect can be determined byvarious types of assays or measure methods thereby differentiating orranking the chelating agents according to their chelating effect (orstrength).

In an assay the chelating agents may be characterized by their abilityto reduce the concentration of free calcium ions (Ca²⁺) from 2.0 mM to0.10 mM or less at pH 8.0, e.g. by using a test based on the methoddescribed by M. K. Nagarajan et al., JAOCS, Vol. 61, no. 9 (September1984), pp. 1475-1478.

For reference, a chelator having the same ability to reduce theconcentration of free calcium ions (Ca²⁺) from 2.0 mM to 0.10 mM at pHas EDTA at equal concentrations of the chelator are said to be strongchelators.

The composition of the present invention may be in any convenient form,e.g., a bar, a homogenous tablet, a tablet having two or more layers, apouch having one or more compartments, a regular or compact powder, agranule, a paste, a gel, or a regular, compact or concentrated liquid.There are a number of detergent formulation forms such as layers (sameor different phases), pouches, as well as forms for machine dosing unit.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition from the pouchprior to water contact. The pouch is made from water soluble film whichencloses an inner volume. Said inner volume can be divided intocompartments of the pouch. Preferred films are polymeric materialspreferably polymers which are formed into a film or sheet. Preferredpolymers, copolymers or derivatives thereof are selected polyacrylates,and water soluble acrylate copolymers, methyl cellulose, carboxy methylcellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, mostpreferably polyvinyl alcohol copolymers and, hydroxyprpyl methylcellulose (HPMC). Preferably the level of polymer in the film forexample PVA is at least about 60%. Preferred average molecular weightwill typically be about 20,000 to about 150,000. Films can also be ofblend compositions comprising hydrolytically degradable and watersoluble polymer blends such as polyactide and polyvinyl alcohol (knownunder the Trade reference M8630 as sold by Chris Craft In. Prod. OfGary, Ind., US) plus plasticisers like glycerol, ethylene glycerol,Propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids. Ref: (US2009/0011970A1).

Detergent ingredients may be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components may beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent. A liquid or geldetergent may be non-aqueous.

Another form of composition is in the form of a soap bar, such as alaundry soap bar, and may be used for hand washing laundry, fabricsand/or textiles. The term “soap bar” as used herein, refers to includeslaundry bars, soap bars, combo bars, syndet bars and detergent bars. Thetypes of bar usually differ in the type of surfactant they contain, andthe term laundry soap bar includes those containing soaps from fattyacids and/or synthetic soaps. The laundry soap bar has a physical formwhich is solid and not a liquid, gel or a powder at room temperature.The term “solid” as used herein, refers to a physical form which doesnot significantly change over time, i.e. if a solid object (e.g. laundrysoap bar) is placed inside a container, the solid object does not changeto fill the container it is placed in. The bar is a solid typically inbar form but can be in other solid shapes such as round or oval.

The soap bar may also comprise complexing agents like EDTA and HEDP,perfumes and/or different type of fillers, surfactants e.g. anionicsynthetic surfactants, builders, polymeric soil release agents,detergent chelators, stabilizing agents, fillers, dyes, colorants, dyetransfer inhibitors, alkoxylated polycarbonates, suds suppressers,structurants, binders, leaching agents, bleaching activators, clay soilremoval agents, anti-redeposition agents, polymeric dispersing agents,brighteners, fabric softeners, perfumes and/or other compounds known inthe art.

The soap bar may be processed in conventional laundry soap bar makingequipment such as but not limited to: mixers, plodders, e.g. a two stagevacuum plodder, extruders, cutters, logo-stampers, cooling tunnels andwrappers. The invention is not limited to preparing the soap bars by anysingle method. The premix of the invention may be added to the soap atdifferent stages of the process. For example, the premix comprising asoap, an enzyme, optionally one or more additional enzymes, a proteaseinhibitor, and a salt of a monovalent cation and an organic anion may beprepared and the mixture may then plodded. The enzyme and optionaladditional enzymes may be added at the same time as an enzyme inhibitor,e.g. a protease inhibitor, for example in liquid form. Besides themixing step and the plodding step, the process may further comprise thesteps of milling, extruding, cutting, stamping, cooling and/or wrapping.

Uses

The present invention further relates to the use of a variant accordingto the present invention in a cleaning process such as laundry or hardsurface cleaning including automated dish wash and industrial cleaning.The soils and stains that are important for cleaning are composed ofmany different substances, and a range of different enzymes, all withdifferent substrate specificities, have been developed for use indetergents both in relation to laundry and hard surface cleaning, suchas dishwashing. These enzymes are considered to provide an enzymedetergency benefit, since they specifically improve stain removal in thecleaning process that they are used in, compared to the same processwithout enzymes. Stain removing enzymes that are known in the artinclude enzymes such as proteases, amylases, lipases, cutinases,cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases,xanthanases, peroxidaes, haloperoxygenases, catalases and mannanases.

In one embodiment, the invention relates the use of variants of thepresent invention in detergent compositions, for use in cleaninghard-surfaces, such as dish wash, or in laundering or for stain removal.In another embodiment, the invention relates to the use of analpha-amylase variant according to the invention in a cleaning processsuch as laundry or hard surface cleaning including, but not limited to,dish wash and industrial cleaning. Thus, in one embodiment, theinvention relates to the use of a variant comprising an alteration inone or more positions corresponding to the positions H1, H2, D3, G4, T5,G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22,H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36,A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50,T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69,N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85,E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100,V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117,Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129,E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141,G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155,K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167,D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179,F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193,E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206,M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220,G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233,V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251,H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263,V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277,Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291,P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303,S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316,V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331,T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349,P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365,V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379,P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392,Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407,W408, D409, V410, I411, T414, R415, E416, N418, A419, S420, H421, P422,S424, G425, L426, A427, T428, I429, M430, S431, D432, G435, G436, S437,K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448, V450, W451,H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466,D467, W469, H471, F473, N475, G476, G477, V481, and W482; wherein thepositions correspond to amino acid positions in the amino acid sequenceset forth in SEQ ID NO: 3 in a cleaning process such as laundry or hardsurface cleaning including dish wash and industrial cleaning.

In a particular embodiment, the invention relates to the use of avariant comprising a) a substitution and/or deletion of two, three orfour positions in the parent alpha-amylase said positions correspondingto positions R181, G182, D183, and G184 of the mature polypeptide of SEQID NO: 4; and

b) a substitution in one or more positions corresponding to positionsH1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17,P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32,N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46,A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63,Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81,K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96,I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113,T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125,N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137,E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150,Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163,G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175,R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189,E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202,Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215,L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228,N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247,D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259,N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273,A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285,M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299,A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312,L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326,T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345,G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360,Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374,P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388,L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403,Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E416, N418,A419, S420, H421, P422, S424, G425, L426, A427, T428, I429, M430, S431,D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446,A447, G448, V450, W451, H452, D453, M454, T455, G456, N457, S459, G460,T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481,and W482; wherein the positions correspond to amino acid positions inthe amino acid sequence set forth in SEQ ID NO: 3, in a cleaning processsuch as laundry or hard surface cleaning including dish wash andindustrial cleaning.

In one embodiment of the invention relates the use of a compositionaccording to the invention comprising a variant of the present inventiontogether with one or more surfactants and optionally one or moredetergent components, selected from the list comprising of hydrotropes,builders and co-builders, bleaching systems, polymers, fabric hueingagents and adjunct materials, or any mixture thereof in detergentcompositions and in detergent applications.

A further embodiment is the use of the composition according to theinvention comprising a variant of the present invention together withone or more surfactants, and one or more additional enzymes selectedfrom the group comprising of proteases, lipases, cutinases, cellulases,endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases,peroxidaes, haloperoxygenases, catalases and mannanases, or any mixturethereof in detergent compositions and in detergent applications.

In another aspect, the invention relates to a laundering process whichmay be for household laundering as well as industrial laundering.Furthermore, the invention relates to a process for the laundering oftextiles (e.g. fabrics, garments, cloths etc.) where the processcomprises treating the textile with a washing solution containing adetergent composition and an alpha-amylase of the present invention. Thelaundering can for example be carried out using a household or anindustrial washing machine or be carried out by hand using a detergentcomposition containing a glucoamylase of the invention.

In another aspect, the invention relates to a dish wash process whichmay be for household dish wash as well as industrial dish wash. The term“dish wash” as used herein, refers to both manual dish wash andautomated dish wash. Furthermore, the invention relates to a process forthe washing of hard surfaces (e.g. cutlery such as knives, forks,spoons; crockery such as plates, glasses, bowls; and pans) where theprocess comprises treating the hard surface with a washing solutioncontaining a detergent composition and an alpha-amylase variant of thepresent invention. The hard surface washing can for example be carriedout using a household or an industrial dishwasher or be carried out byhand using a detergent composition containing an alpha-amylase of theinvention, optionally together with one or more further enzymes selectedfrom the group comprising of proteases, amylases, lipases, cutinases,cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases,xanthanases, peroxidaes, haloperoxygenases, catalases, mannanases, orany mixture thereof.

In a further aspect, the invention relates to a method for removing astain from a surface comprising contacting the surface with acomposition comprising an alpha-amylase of the present inventiontogether with one or more surfactants and optionally one or moredetergent components selected from the list comprising of hydrotropes,builders and co-builders, bleaching systems, polymers, fabric hueingagents and adjunct materials, or any mixture thereof in detergentcompositions and in detergent applications. A further aspect is a methodfor removing a stain from a surface comprising contacting the surfacewith a composition comprising an alpha-amylase variant of the presentinvention together with one or more surfactants, one or more additionalenzymes selected from the group comprising of proteases, lipases,cutinases, cellulases, endoglucanases, xyloglucanases, pectinases,pectin lyases, xanthanases, peroxidaes, haloperoxygenases, catalases andmannanases, or any mixture thereof in detergent compositions and indetergent applications.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1 Generation of Variants According to the Invention

The variants of the present invention have been generated bysite-directed mutagenesis. Genomic DNA prepared from the organismcontaining amylase gene at the Pel locus was used as template forgenerating the site-directed mutants.

Mutagenic forward primer and PnMi4490 (CAATCCAAGAGAACCCTGATACGGATG—SEQID NO: 5) reverse primer was used to generate a ˜3.8 kb fragment. Thisfragment was used as a megaprimer along with PnMi4491(CGGAACGCCTGGCTGACAACACG—SEQ ID NO: 6) forward primer to get 6 kbinsertion cassette. To enable integration in the Pel locus by doublecross-over upon transformation, along with the amylase and cat genes,the cassette contained upstream and downstream Pel sequences at theends. Selection was done on LB Agar containing chloramphenicol and themutation was confirmed by DNA sequencing of amylase gene.

A library of variants were generated with a substitution in differentpositions selected from the following positions; H1, H2, D3, G4, T5, G7,T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23,W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37,G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51,S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70,Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86,R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103,H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118,A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130,V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142,F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156,W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168,Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180,R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194,N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208,D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221,V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238,H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252,V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264,Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278,L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292,L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304,G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318,Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334,P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350,L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366,F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380,S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394,Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408,D409, V410, I411, T414, R415, E416, N418, A419, S420, H421, P422, S424,G425, L426, A427, T428, I429, M430, S431, D432, G435, G436, S437, K438,W439, M440, Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452,D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467,W469, H471, F473, N475, G476, G477, V481, and W482; wherein thepositions correspond to amino acid positions in the amino acid sequenceset forth in SEQ ID NO: 3.

TABLE 1 Variants according to the invention H1V Q98T Y178R L275R P375VH1G Q98F Y178K L275K P375R H1S Q98W Y178W E276R S376A H1C Q98P K179LE276L S376N H1L Q98H K179C N277D S376P H2L V99I K179E Y278G S376R H2TV99L K179S Y278S S376W H2M V99S K179W L279Q S376L H2E V99T K179A L279VS376V H2P V99G K179G L279G S376I H2G V99E K179T L279M S376Q D3C Y100WK179F S280C S376F D3A Y100V F180A S280R S376E D3R Y100F F180L S280ES376T D3Y V103G R181K S280D D377C D3W V103A R181P K281L D377L D3S V103SR181S K281A D377V D3F V103T R181A K281W D377S G4Q H107Q R181V K281GD377G G4D H107M R181Q T282G D377T G4R K108V R181G T282A V379L T5L G109AR181E T282C V379F T5Q G109S R181D T282R V379P T5G G109D G184L T282IV379S T5E G109F G184R N283G P380L T5V G110C G184V N283E P380V T5F A111SG184T N283Q P380G T5W D112E G184D N283V P380E G7F D112V K185A N283AP380A G7R D112F K185D W284T P380I G7V D112C K185N W284I P380Y G7E D112KK185G W284S S381N G7E F113M K185V W284E S381V G7D F113V A186N W284CS381L G7L F113L A186E W284V S381A G7A F113C A186S W284A S381I G7S F113EA186C T285E Y382I G7N F113D A186D T285F Y382A T8A F113K A186T T285SY382E T8S F113Q A186Q T285W Y382V I9C F113Y A186V T285M Y382L I9M T114SA186H T285P Y382K I9L T114V W187G M286L Y382R M10L T114L D188Q M286DR383G Q11C E115C D188G M286C R383S Q11L E115A D188R S287V R383P Q11VR116N D188M S287Q R383V Q11M R116L D188L S287T Q384A Y12V R116I D188VS287E Q384P Y12A R116Q W189R S287P Q384L Y12F R116D W189A S287L Q384HF13Y R116T W189G S287W Q384R F13I R116G W189C S287C Q384Y E14L R116EE190D A288L Q384S E14M R116S E190N A288Y Q384V E14S R116C E190A A288VQ384T E14Q R116V V191M V291T Q384G E14F R116F V191A P292A Q384E W15GR116K D192E P292Q I386T W15F R116H D192Q P292L I386L W15S R116W D192SP292R I386M W15I V117C T193S P292G I386W W15H V117L T193K L293A I386VN16G V117S T193V L293E D387H N16Q V117W T193C L293T D387Q N16R V117AT193P L293G D387V V17D V117G T193D L293Y D387L V17G V117E T193R L293QP388G V17E V117I T193G H294G P388T V17Q V117M E194S H294A P388M V17AQ118V E194D H294W P388W V17T Q118G E194L H294Q P388V V17L Q118E E194QH294S P388S P18L Q118F N195R Y295H P388R N19Y Q118R N195A Y295S P388KN19T A119Y N195T Y295I P388L N19G A119W N195F Y295A L389S N19S V120IG196A Y295F L389H N19V V120D G196V N296A L389A D20R V120L G196P N296YL389V Q22N V120C N197G N296M L389F Q22C V120A N197V N296F L389M Q22SV120N N197Y N296S L390V Q22M E121P N197C N296K L390S Q22T E121G N197DN296H L390F Q22E E121V Y198W L297S L390I Q22A E121M Y198A L297W L390GQ22Y E121D Y198G L297H L390T H23W V122E D199G Y298V L390N W24H V122PD199S Y298P L390H W24F V122L D199A Q299S L390C N25V V122I Y200W Q299LK391L N25S V122F Y200F Q299R K391Y N25G N123G Y200C Q299I K391A N25EN123A Y200V Q299A K391Q N25E N123E Y200T Q299H A392I N25R N123W Y200LQ299P A392M N25L N123T Y200A A300V A392T N25T N123P Y200G A300C Q394SR26G N123K Y200S S301A Q394R R26D N123V M202T S301R Q394N R26M P124RM202L S301Q Q394L R26I P124G Y203F N302I Q394T R26S Q125E Y203G N302TQ394M R26L Q125T Y203C N302M Q395W R26W N126E Y203I N302S Q395E R26AN126G Y203Q S303I Q395K R26F N126A Y203H S303E Q395N R26E N126R Y203WS303A Q395L L27A N126T Y203T S303M Q395R L27M R127G Y203L S304I Q395VH28A R127S Y203M S304P Q395S H28E R127C Y203S S304G Q395M H28I R127WY203V S304L Y396T H28R R127L A204P S304F Y396L H28M R127V A204R S304AY396R H28W R127F A204C S304M Y396W H28K R127Y A204H G305V A397C N29CN128S A204W G305L A397G N29G N128E A204F G305S A397S N29L N128C A204LG305Q Y398S N29K N128P A204Y G305F Y398C N29V N128G A204D G305I Y398NN29W N128A A204G N306V Y398K N29A Q129L D205C N306L R400L N30S Q129CD205R N306D R400C N30H Q129E D205A N306S R400K N30L E130C D205L N306TR400S N30T V131L V206H Y307F R400M N30E V131E V206Q Y307W R400E N30RV131I V206P M309L R400I A31W S132A V206A R310A H402L A31V S132H V206SR310T H402V A31S S132P V206K N311L H402E A31G S132C V206L N311R H402MA31R S132E V206I N311C H402A Q32D S132D V206M N311Y H402S Q32G S132VV206F L312F H402P Q32L S132Q V206Y L312V H402Y Q32A S132N M208R L312IH402D N33F G133S M208P L313S H402R N33A G133T M208L L313M H402T N33RG133L M208C L313I D403E N33D G133A D209C N314Y D403P L34F G133V D209SN314F D403Q L34Y G133I D209M N314D D403I K35L G133E D209E G315I Y404KK35E G133D D209H G315V Y404F K35W G133R D209F G315T Y404W K35Q T134VD209Y G315R F405G N36R T134D D209V G315F F405C N36Q T134E D209Q T316KF405V N36A Y135L D209L T316S F405I N36E Y135V D209I T316C F405P N36VY135S H210F T316G F405W N36P Y135R H210V V318C F405A N36W Y135T H210AV318L D406E N36T Y135W H210W V318E D406V A37R Y135K H210L V318A D406TA37G Y135G H210G Q319L D406M A37T Y135P H210D Q319I D406S A37S Y135MH210E Q319A H407C A37E Y135D H210Q Q319P H407D G38N Y1315H H210N Q319HW408R G38M Y135F P211G Q319D W408P G38Y Y135E P211W Q319F W408C G38KQ136E P211C R320V W408Y I39L Q136W E212G R320L W408G I39M Q136G E212FR320C W408D I39V I137A E212M R320A D409G T40Q I137S E212D R320S V410IT40D I137G E212I H321T V410Q T40A 1137V E212Y H321E V410L T40E E138AE212R H321V V410E T40S E138Y E212C H321C I411A A41E E138T E212K H321GI411L A41N E138L V213C H321R I411V A41M A139M V213M H321S T414A A41QA139S V213T P322E T414V A41D A139G V213G P322C R415F I42V A139V V213WS323N R415L I42A A139T V213R A325V R415A I42L A139P V213F A325S R415QW43R W140Y I214P V326I E416C I44T W140S 1214V V326G E416V I44L W140HN215D V326A E416L I44V W140A N215C V326C E416A P45A T141S N215V V326TE416Q P46G T141M N215T T327V N418L A47S T141C L217V T327L N418M A47GT141G L217C T327S N418A A47T G142D L217P T327Q N418C W48G G142E L217TT327E N418G W48L G142R L217G F328M N418R W48C G142A L217S F328C N418FW48I F143M L217A V329A A419M W48M F143L L217W V329S A419E W48P F143IN218S V329C A419C W48Q N144C N218D V329I A419G W48H N144M N218R N331CA419V W48A N144K N218L N331V A419L W48Y N144G N218M N331I A419Q W48FN144R R219L T334A A419R K49Q F145C R219H T334S S420E G50P F145S R219WP336M S420T G50S F145A R219S P336E S420V G50N F145N R219I P336D S420MT51S F145Q R219A P336L H421V T51G F145T R219M P336R H421C T51P F145HR219T P336W H421A T51L P146S R219E P336S H421R T51C P146V R219Y P336KH421T T51A P146L R219D G337T P422Y S52M P146C R219C G337C P422H S52VP146M W220L G337V P422D S52I P146T W220I G337M P422R S52L G147V W220PG337F S424C S52C G147P W220E E338T S424A S52F G147D W220R E338A S424LS52A G147S W220A E338H G425A S52D G147I W220S E341C G425V S52K G147LG221S E341D L426S S52N G147C G221A E341I L426A S52E G149C V222E E341PA427V S52T G149Y V222F E341M A427S Q53G G149L V222C E341F T428L Q53VG149A V222L E341K T428N Q53L N150C W223Y E341S T428A Q53M N150D W223FE341T T428G N54G N150E W223L S342G I429V N54K Q151L Y224L S342T I429AN54V Q151T A225K S342A M430L N54T Q151C N226C V344S M430R N54L Q151IN226Q V344L M430A N54I S153C N226M Q345K M430T N54S S153G N226E Q345CS431A D55V S153T N226K Q345V S431T D55I S154M N226R Q345L S431F D55GS154E T227S Q345I D432Q V56L S154A T227L Q345W D432P V56M S154V T227MQ345N G435N V56T F155H T227C G346H G435E G59E F155T T227Y G346A G435MG59A F155L T227R G346R G435I G59S F155W L228A G346L G435S A60S K156YL228F G346N G436M A60W K156V L228M G346Q G436E A60L K156A N229T G346VG436L A60G K156F N229S G346S G436T A60V K156D L230S W347L S437G A60FK156Q L230A W347R S437Q A60T K156L L230T W347V S437L Y61F K156S L230VW347C K438T L63V K156W D231E F348Q W439E L63P K156P D231C F348S W439CL63I K156E D231G F348C W439H L63H K156C D231P F348P W439A L63K K156GF233A F348M W439R L63C K156N F233L K349G W439T L63G K156T F233T K349AW439N L63A W157L F233P K349Y W439R Y64W R158M F233M K349L W439N Y64SR158A V238C K349S M440L Y64V R158G V238W P350S Y441N Y64I R158Q V238AP350L Y441H Y64H R158S H240W P350G Y441Q Y64M R158W I241L P350Q Y441KY64C R158L K242A P350D V442I Y64R R158T K242C P350E R444A Y64G R158VK242Y L351W R444S Y64L R158E K242P L351M R444L Y64A R158Y K242W L351SR444V L66M W159N K242G L351H R444P E68D W159G K242L L351Q Q445L E68SW159T K242R A352S Q445S E68M W159C K242S A352C Q445C E68C W159E K242EA354C Q445E E68T W159D K242Q T355V Q445R E68V W159S K242H T355A K446GE68Q W159H S244Q T355L K446Q E68Y Y160Q F245C T355Y K446T F69H Y160MF245V T355F K446P F69W Y160T F245D L357C A447G N70G Y160A F245G L357TA447Q N70A Y160S F245A L357S A447E N70T Y160V M246L L357H A447T N70SY160G M246F T358V A447R N70E Y160I M246V T358F A447S Q71N Y160L M246IT358S G448S Q71E Y160E R247Y T358L G448R Q71D Y160W R247G E360S V450TQ71I Y160R R247M E360D V450A Q71V Y160H D248G E360C V450Q K72Y H161CD248R E360G V450R K72R H161N W249L E360A W451L G73V F162M W249F E360MH452A G73L F162L L250C Q361V H452T G73E F162Y L250W Q361P H452C G73CF162I L250T Q361R H452G T74V D163S L250A Q361A H452Y T74L D163K L250FQ361E H452R T74N D163A L250Y Q361G H452V V75T D163R G251K Q361D D453EV75G D163L G251M Q361S M454R V75E G164S G251Q G362Y M454L R76S G164TH252C G362R M454V R76G G164C H252E G362C M454Q R76A T165L H252W G362NM454S R76V T165M H252T G362M T455L T77A T165S H252F G362V T455S T77ST165G H252V Y363L G456Y T77V T165I V253A Y363Q G456V K78L T165V V253LY363I G456R K78T T165E V253S Y363A G456K K78A D166T V253W Y363R G456LT81G D166S R254Q Y363V N457V T81W D166N R254S Q365D N457I T81L W167YG255C Q365M N457G K82A W167G G255E Q365L N457R K82G W167D G255L Q365TS459E A83G W167F Q256P Q365C S459Q A83E D168M T257V Q365S S459V A83QD168S G258P V366L S459C A83R D168G G258V V366A S459P A83S Q169L G258CV366I S459M A83F Q169A G258I V366C S459T A83V Q169S G258L F367L S459RE84F Q169V G258F F367S G460S E84D Q169F G258D F367A G460D E84M Q169EK259L Y368M G460E E84W S170A K259V Y368T T463C L85V S170V K259Q Y368HT463V E86L S170Q K259I Y368I T463L E86D S170W K259A Y368L T463D E86WS170G K259S Y368A T463H E86R S170F K259D Y368S T463M R87M S170T K259CY368Q T463A R87A S170L N260W Y368V T463Q R87E R171F N260E G369K I464VA88S R171S N260D G369S N465G A88G R171K N260C G369A N465L A88V R171AN260S G369E N465R I89A R171V N260R G369R N465Q I89H R171M N260G G369IQ466L I89P Q172L L261G G369L Q466C I89L Q172E L261D D370A Q466E R90LQ172S L261T D370T D467C R90G Q172C F262Q Y372A D467V S91A Q172R F262LY372S W469M S91E Q172G F262E Y372I W469E S91G Q172W F262R Y372L W469TS91V Q172K F262P Y372G W469N S91T L173I F262V G373C H471Q L92C L173GA263L G373S F473Y L92F L173N A263C G373T F473C K93W L173M V264T G373QF473R K93G L173S V264L G373W F473E K93L L173Y Y267V G373R F473G N95LL173V Y267L I374Y F473T N95E L173A W268V I374S F473V N95W L173F W268YI374M N475T N95C A174V K269W I374L N475H N95S A174G K269L P375L N475GN95G A174L K269Q P375A G476F N95D A174M K269R P375T G476Y N95R A174KN270D P375E G476K G96Q A174S N270A P375S G477Q G96R N175L N270M G477FI97M N175V N270G G477K I97V N175I D271E G477L I97L N175G D271V V481QQ98V N175S D271C V481S Q98L R176E L272M W482F Q98C R176W G273W Q98MR176G G273R R176M G273V R176Y G273E R176M A274S R176T A274D R176K A274EI177L L275G I177S L275E I177G L275A Y178V

Example 2 Specific Activity of Variants

In order to determine whether the variants generated as described andlisted in Example 2 have a maintained or even improved activity, thevariants were evaluated by the Phadebas assay. The following detergentcompositions were prepared;

Preparation of Model X (0.175%):

1:2 molar ratio of CaCl2 and MgCl2 stock solution with 6000 dH (waterhardness).

104.9 g of CaCl₂.2H₂O (0.713M) was weighed into 1 liter bottle and tothis 500 ml of type I water was added and stirred well. To this 72.5 gof MgCl2.6H2O (0.357M) was weighed and added, dissolved well and thefinal volume was made upto 1000 ml with type I water.

0.535 M Solution of NaHCO₃

44.9 g of Sodium Hydrogen carbonate was dissolved in 100 ml of type Iwater.

Model X Detergent with X Ionics with a Water Hardness of 12 (12° dH)

1.75 g of Model X detergent (as described above) was weighed andtransferred into 1 litre bottle and to this 800 ml of type I water wasadded and mixed well. To this 35 mg of X-ionics was added and mixedwell. To adjust the water hardness to 12° dH, 2 ml of 1:2 molar ratio ofCaCl₂ and MgCl₂ stock solution with 6000° dH, 6 ml of 0.535 Molarsolution of NaHCO₃ was added and mixed well. Finally the volume was madeup to 1000 ml and the mixture was stirred for 10 min.

Preparation of Model A (0.33%):

-   4:1 Molar Ratio of CaCl2 and MgCl2 Stock Solution with 6000 dH    (Water Hardness)

125.8 g of CaCl2.2H2O was weighed into 1 liter bottle and to this 500 mlof type I water was added and stirred well. To this 43.8 g of MgCl2.6H2Owas weighed and added and dissolved well and the final volume was madeup to 1000 ml with type I water.

0.535 M Solution of NaHCO₃

44.9 g of Sodium Hydrogen carbonate was dissolved in 100 ml of type Iwater.

Model A Detergent with a Water Hardness of 15 (15° dH)

3.335 g of Model A detergent was weighed and transferred into 1 litrebottle and to this 865 ml of type I water was added and mixed well. Tothis 7.5 ml of 0.535M NaHCO₃ was added, mixed well and made up thevolume to 1 liter with type 1 water. To adjust the water hardness to 15°dH 2.5 ml of 4:1 molar ratio of CaCl₂.2H₂O and MgCl₂.6H₂O stock solutionwith 6000° dH was added and the mixture was stirred for 15 min.

Substrate: Phadebas Tablets (Magle Life Sciences)

1 tablet was suspended in 10 ml of the detergent solutions.

Buffer: 100 mM MOPS Buffer pH 8

Experimental Procedure

Preparation of the Mother Plates:

Colonies were picked from the transformed plate by colony picker(KBiosystems) and inoculated in 96-well culture plate comprising TBGlymedia for growth. The cultures were grown for 3 days at 37° C. and thesupernatant was recovered from the plates by centrifugation.

Preparation of the Substrate Plates:

The substrate solution was prepared by dissolving 1 tablet of phadebasin 10 ml of Model X/Model A detergent and 180 ul of the same wasdispensed into 96 Well micro titer plate using multidrop instrument withconstant stirring.

The culture supernatant was diluted to 100× with buffer and 20 ul of thediluted culture was added to the 180 ul of pre-dispensed substrate plateand mixed well. The plate was incubated for 20 min at 25 C with shaking(900 rpm). After the incubation the plate was allowed to settle for 5mins. 50 ul of the supernatant was transferred into 384 well plates andthe absorbance was measured at 620 nm. The concentration of theexpressed enzyme was determined by ELISA using specific antibodies. Thespecific activity was calculated by taking the ratio of the activity byconcentration and the hits were identified as anything higher than thespecific activity of the parent alpha-amylase. The Improvement Factor(IF) was calculated as:[Specific Activity of variant]/[Specific Activity of parentalpha-amylase].

TABLE 2 The specific activity of the variants according to the inventionobtained by the method described above are the following (amino acidsubstitutions refer to SEQ ID NO: 3); Substi- Substi- tution Model AModel X tution Model A Model X H1V 1.5 1.3 V206H 1.3 1.2 H1G 1.5 1.4V206Q 1.3 1.2 H1S 1.5 1.3 V206P 1.4 1.3 H1C 1.6 1.5 M208R 1.1 1.2 H2L1.2 1.3 M208P 1.1 1.1 H2T 1.3 1.1 M208L 1.1 1.1 H2M 1.3 1.2 M208C 1.21.2 H2E 1.4 1.2 D209C 1.1 0.9 H2P 1.4 1.3 D209S 1.3 1.0 H2G 1.4 1.5D209M 1.3 1.0 D3C 1.1 1.2 D209E 1.3 1.3 D3A 1.3 1.5 D209H 1.3 1.3 G4Q1.3 1.3 D209F 1.3 0.8 G4D 1.4 1.4 D209Y 1.3 1.0 G4R 1.5 D209V 1.4 1.1T5L 1.1 1.3 H210F 0.5 1.6 T5Q 1.2 1.4 H210V 1.5 1.3 T5G 1.2 1.3 H210A1.5 1.6 T5E 1.4 1.4 H210W 1.6 1.7 T5V 1.4 1.4 H210L 1.6 1.8 G7F 1.4 1.5H210G 1.7 1.7 G7R 1.5 1.5 H210D 1.8 2.0 G7V 1.8 1.6 H210E 2.0 2.0 G7E1.8 1.6 P211G 1.3 1.2 G7E 1.9 1.5 P211W 1.3 1.2 G7D 2.1 1.8 P211C 1.41.2 T8A 1.0 1.2 E212G 1.2 1.4 T8S 1.2 1.2 E212F 1.2 1.4 I9C 1.0 1.3E212M 1.4 1.5 I9M 1.3 1.4 E212D 1.4 1.4 I9L 1.3 1.3 E212I 1.5 1.5 M10L1.1 1.1 V213C 1.1 1.3 Q11C 1.1 1.3 V213M 1.1 1.2 Q11L 1.2 1.2 V213T 1.11.3 Q11V 1.5 1.4 V213G 1.1 1.1 Y12V 1.2 1.1 V213W 1.2 1.2 Y12A 1.3 1.3I214P 1.3 1.2 Y12F 1.3 1.3 N215D 1.6 1.5 F13Y 1.4 1.3 N215C 1.7 1.4 F13I1.7 1.6 N215V 1.7 1.6 E14L 1.9 1.0 L217V 1.1 1.3 E14M 2.0 1.4 L217C 1.31.4 E14S 2.0 1.2 L217P 1.4 1.4 W15G 1.5 0.5 L217T 1.5 1.3 W15F 1.6 1.1L217G 1.5 1.3 W15S 2.0 0.5 L217S 1.7 1.2 N16G 1.5 1.4 N218S 1.1 1.2 N16Q1.6 1.7 R219L 1.5 1.3 P18L 1.5 1.2 R219H 1.5 1.4 N19Y 1.5 1.4 R219W 1.51.3 N19T 1.5 1.6 R219S 1.5 1.3 N19G 1.6 1.6 R219I 1.5 1.5 N19S 1.7 1.6R219A 1.5 1.3 N19V 1.7 1.6 R219M 1.5 1.4 D20R 2.1 R219T 1.5 1.4 Q22N 1.31.4 R219E 1.5 1.4 Q22C 1.4 1.5 R219Y 1.7 1.6 Q22S 1.4 1.4 R219D 1.7 1.6Q22M 1.5 1.4 R219C 1.7 1.6 Q22T 1.5 1.3 V222E 1.3 1.2 Q22E 1.5 1.3 V222F1.3 1.2 Q22A 2.2 1.5 V222C 1.3 1.2 W24H 0.9 1.3 W223Y 1.7 1.6 W24F 1.31.5 Y224L 1.1 1.3 N25V 1.4 1.3 A225K 1.2 1.3 N25S 1.4 1.3 N226C 1.3 1.4N25G 1.4 1.4 N226Q 1.4 1.5 N25E 1.4 1.5 T227S 1.2 1.2 N25E 1.5 1.4 T227L1.2 1.2 R26G 1.5 1.5 T227M 1.2 1.1 R26D 1.5 1.4 T227C 1.2 1.2 R26M 1.51.3 L228A 1.2 1.2 R26I 1.5 1.4 L228F 1.3 1.2 R26S 1.5 1.4 N229T 1.1 1.3R26L 1.6 1.5 N229S 1.2 1.3 R26W 1.6 1.5 L230S 1.2 1.1 R26A 1.6 1.6 L230A1.3 1.2 R26F 1.7 1.5 L230T 1.3 1.2 L27A 1.2 1.2 L230V 1.4 1.4 L27M 1.31.2 D231E 1.0 H28A 1.2 1.3 D231C 1.0 H28E 1.3 1.2 D231G 1.3 H28I 1.3 1.3F233A 1.3 1.3 N29C 1.3 1.3 F233L 1.3 1.3 N29G 1.6 1.7 V238C 1.7 1.5 N29L1.6 1.5 V238W 2.1 2.0 N30S 1.2 1.3 I241L 1.5 1.4 N30H 1.3 1.2 K242A 1.51.7 N30L 1.4 1.3 K242C 1.5 1.5 N30T 1.4 1.2 K242Y 1.5 1.5 N30E 1.6 1.3K242P 1.5 1.4 A31W 1.1 1.4 K242W 1.6 1.4 A31V 1.4 1.3 K242G 1.6 1.5 A31S1.6 1.6 K242L 1.6 1.5 Q32D 1.4 1.3 K242R 1.8 1.9 Q32G 1.5 1.5 K242S 2.22.4 Q32L 1.6 1.5 K242E 2.9 3.2 Q32A 1.6 1.5 F245C 1.7 1.5 N33F 0.9 1.0F245V 1.7 1.5 N33A 0.9 1.1 F245D 1.9 1.8 N33R 0.9 1.0 F245G 2.0 1.9 L34F1.2 1.3 M246L 1.2 1.1 L34Y 1.4 1.4 M246F 1.2 0.8 K35L 1.3 1.1 W249L 1.31.3 K35E 1.3 1.1 L250C 1.1 1 K35W 1.3 1.0 L250W 1.1 1.1 K35Q 1.3 1.0L250T 1.2 1.1 N36R 1.2 1.1 L250A 1.2 1.2 N36Q 1.3 1 H252C 1.2 1.2 N36A1.3 1.3 H252E 1.2 1.3 N36E 1.3 1.4 H252W 1.2 1.1 A37R 1.0 1.3 V253A 1.11.1 A37G 1.1 1.4 V253L 1.1 1 A37T 1.3 1.3 V253S 1.1 0.9 A37S 1.3 1.2V253W 1.1 1 G38N 1.0 1.1 R254Q 1.2 1.4 I39L 1.2 1.1 R254S 1.3 1.5 I39M1.2 1.2 G255C 1.1 1 T40Q 1.1 1.1 G255E 1.2 1.2 T40D 1.1 1.0 G255L 1.21.1 T40A 1.2 1.0 Q256P 1.1 1.3 T40E 1.2 0.9 T257V 1.2 1.1 A41E 1.3 1.3G258P 1.3 1.3 A41N 1.3 1.2 G258V 1.3 1.3 A41M 1.3 1.2 G258C 1.3 1.2 A41Q1.4 1.4 G258I 1.3 1.3 A41D 1.4 1.3 G258L 1.9 1.9 I42V 1.1 1.3 K259L 1.31.2 I42A 1.1 1.2 K259V 1.3 1.2 W43R 1.2 1.3 K259Q 1.3 1.2 I44T 1.2 1.1K259I 1.4 1.2 P45A 1.3 1.4 N260W 1.3 1.3 P46G 1.9 1.6 N260E 1.3 1.2 A47S1.0 1.0 N260D 1.4 1.4 A47G 1.1 1.2 N260C 1.4 1.3 W48G 1.3 1.4 L261G 1.31.3 W48L 1.3 1.3 L261D 1.3 1.3 W48C 1.4 1.5 F262Q 1.5 1.8 W48I 1.4 1.4F262L 1.5 1.5 W48M 1.4 1.4 F262E 1.6 1.6 W48P 1.4 1.4 F262R 1.6 1.6 W48Q1.5 1.4 F262P 1.8 2.0 G50P 1.5 1.3 F262V 1.9 1.8 G50S 1.5 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P388V 1.3 1.3 W159G 1.6 1.4 L389S 1.2 1.3 W159T 1.7 1.7L389H 1.3 1.3 W159C 1.9 2.0 L390V 1.1 1.1 W159E 2.3 2.1 L390S 1.1 1.1W159D 3.0 3.1 L390F 1.1 1.3 Y160Q 1.5 1.5 L390I 1.2 1.2 Y160M 1.5 1.3L390G 1.2 1.2 Y160T 1.5 1.5 K391L 1.1 1.2 Y160A 1.5 1.3 K391Y 1.2 1.2Y160S 1.7 1.7 K391A 1.2 1.2 Y160V 1.7 1.7 A392I 1.3 1.3 Y160G 1.7 1.5Q394S 1.4 1.4 Y160I 1.7 1.6 Q394R 1.5 1.5 Y160L 1.8 1.7 Q395W 1.1 1.2Y160E 2.2 2.0 Q395E 1.2 1.3 H161C 1.0 1.0 Q395K 1.3 1.3 H161N 1.0 1.0Q395N 1.3 1.3 F162M 1.2 1.1 Q395L 1.3 1.0 F162L 1.3 1.1 Q395R 1.4 1.4F162Y 1.3 1.3 Y396T 1.1 1.1 F162I 1.4 1.3 Y396L 1.2 1.1 D163S 1.1 1.4A397C 1.3 1.3 D163K 1.4 1.4 A397G 1.5 1.5 D163A 1.4 1.3 R400L 1.5 1.2D163R 1.4 1.1 R400C 1.5 1.3 D163L 1.7 1.7 R400K 1.5 1.3 G164S 1.5 1.1R400S 1.5 1.3 G164T 1.7 1.1 R400M 1.6 1.4 G164C 1.7 1.1 H402L 1.2 1.4T165L 1.4 1.3 H402V 1.2 1.3 T165M 1.4 1.4 H402E 1.3 1.2 T165S 1.4 1.3H402M 1.3 1.3 T165G 1.6 1.5 H402A 1.3 1.3 T165I 1.8 1.6 H402S 1.4 1.4D166T 1.7 1.3 H402P 1.5 1.5 D166S 1.8 1.4 H402Y 1.5 1.3 D166N 2.0 1.3D403E 1.0 1.1 W167Y 1.4 1.5 F405G 1.2 1.2 W167G 1.4 1.3 F405C 1.2 1.1W167D 2.0 1.7 F405V 1.2 1.2 D168M 1.3 1.5 F405I 1.3 1.2 D168S 1.3 1.4F405P 1.3 1.2 D168G 1.5 1.6 F405W 1.3 1.2 Q169L 1.6 1.8 D406E 1.3 1.2Q169A 1.6 0.0 H407C 1.3 1.3 S170A 1.5 1.3 H407D 1.5 1.2 S170V 1.7 1.3W408R 1.4 1.2 R171F 1.5 1.4 W408P 1.5 1.4 R171S 1.5 1.4 W408C 1.5 1.4R171K 1.5 1.5 W408Y 1.6 1.5 R171A 1.6 1.4 W408G 1.6 1.3 R171V 1.8 1.8W408D 1.6 1.3 Q172L 1.3 1.3 V410I 1.3 1.3 Q172E 1.4 1.4 V410Q 1.4 1.7Q172S 1.7 1.6 I411A 0.9 1.1 L173I 1.4 1.3 I411L 1.0 1.2 L173G 1.4 1.41411V 1.0 1.3 L173N 1.4 1.4 T414A 1.3 0.5 L173M 1.5 1.5 T414V 1.3 1.2L173S 1.5 1.5 R415F 1.5 1.2 L173Y 1.5 1.4 R415L 1.6 1.4 L173V 1.7 1.6E416C 1.2 1.2 A174V 1.4 1.3 E416V 1.3 1.3 A174G 1.4 1.4 N418L 1.2 1.3N175L 1.3 1.6 N418M 1.4 1.3 N175V 1.4 1.6 N418A 1.4 1.3 N175I 1.5 1.7N418C 1.5 1.4 N175G 1.5 1.7 N418G 1.9 1.8 R176E 1.4 1.4 A419M 1.2 1.2R176W 1.4 1.3 A419E 1.3 1.1 R176G 1.5 1.5 A419C 1.3 1.2 R176M 1.6 1.5S420E 1.1 1.0 I177L 1.4 1.3 S420T 1.1 1.0 I177S 1.4 1.3 S420V 1.1 1.0I177G 1.4 1.5 H421V 1.3 1.3 Y178V 1.5 1.4 H421C 1.4 1.3 Y178R 1.6 1.4H421A 1.4 1.2 Y178K 2.0 1.7 H421R 1.5 1.4 K179L 1.4 1.5 S424C 1.1 0.9K179C 1.6 1.5 S424A 1.1 1.1 K179E 1.6 1.5 G425A 1.3 1.4 K179S 1.7 1.5A427V 1.2 1.1 K179W 1.7 1.5 T428L 1.0 0.9 K179A 1.8 1.7 T428N 1.1 1.1K179G 1.8 1.6 T428A 1.1 1.1 K179T 1.8 1.7 T428G 1.2 1.2 K179F 1.9 1.6I429V 1.2 1.1 F180A 1.3 1.3 I429A 1.2 1.2 F180L 1.4 1.4 M430L 1.1 0.9R181K 1.4 1.4 M430R 1.3 1.1 R181P 1.4 1.5 S431A 1.4 1.4 R181S 1.4 1.5G435N 1.7 1.5 R181A 1.5 1.5 G435E 2.0 1.7 R181V 1.6 1.7 G436M 1.3 1.4R181Q 1.9 1.9 G436E 1.3 1.4 R181G 2.0 1.9 G436L 1.3 1.3 R181E 2.0 1.9G436T 1.4 1.4 G184L 1.2 1.0 S437G 1.1 1.1 G184R 1.4 1.3 S437Q 1.2 1.3G184V 1.4 1.4 S437L 1.2 1.3 K185A 1.9 1.7 K438T 1.1 1.1 K185D 2.0 1.9W439E 1.3 1.2 K185N 2.0 1.7 W439C 1.3 1.3 K185G 2.2 1.9 W439H 1.3 1.4K185V 2.2 1.8 M440L 1.4 1.2 A186S 1.5 1.6 Y441N 1.0 1.0 A186C 1.5 1.5Y441H 1.0 1.0 A186D 1.6 1.5 Y441Q 1.0 1.0 W187G 2.4 1.6 V442I 1.2 1.2D188Q 1.4 1.4 R444A 1.3 1.2 D188G 1.4 1.4 R444S 1.3 1.2 D188R 1.4 1.3R444L 1.3 1.1 D188M 1.6 1.5 R444V 1.4 1.4 D188L 1.6 1.4 R444P 1.5 1.3D188V 1.8 1.6 Q445L 1.2 1.3 W189R 1.6 0.9 Q445S 1.3 1.3 W189A 1.6 1.0Q445C 1.4 1.5 W189G 1.7 0.9 Q445E 2.1 2.0 W189C 1.8 1.1 K446G 1.0 1.0E190D 1.3 1.0 K446Q 1.0 1.0 E190N 1.3 1.0 A447G 1.3 1.3 E190A 1.3 1.2A447Q 1.3 1.3 V191M 1.6 1.3 A447E 1.4 1.5 V191A 1.6 1.1 A447T 1.5 1.4D192E 1.1 1.0 G448S 1.3 1.2 D192Q 1.2 1.0 V450T 1.1 1.1 D192S 1.2 1.0V450A 1.1 1.1 T193S 1.3 1.1 H452A 1.2 1.2 T193K 1.3 1.2 H452T 1.2 1.1T193V 1.3 1.2 D453E 1.1 1.0 T193C 1.4 1.2 M454R 1.2 1.2 T193P 1.4 1.3M454L 1.2 1.0 E194S 1.1 1.0 M454V 1.6 1.1 E194D 1.2 1.0 T455L 1.1 1.0E194L 1.3 1.0 T455S 1.2 1.1 E194Q 1.9 1.6 G456Y 1.2 1.2 N195R 1.3 1.1G456V 1.2 1.2 N195A 1.3 1.1 G456R 1.5 1.6 G196A 1.3 1.2 N457V 1.1 1.2G196V 1.3 1.1 N457I 1.1 1.2 N197G 1.3 1.1 N457G 1.1 1.1 N197V 1.3 1.2N457R 1.2 1.2 N197Y 1.3 1.2 S459E 1.2 0.1 N197C 1.4 1.2 S459Q 1.2 1.2N197D 1.4 1.2 S459V 1.2 1.2 Y198W 1.2 1.1 S459C 1.2 1.1 D199G 1.5 1.5S459P 1.2 1.1 D199S 1.6 1.5 S459M 1.3 1.3 D199A 1.7 1.5 S459T 1.5 1.5Y200W 1.6 1.4 G460S 1.2 1.2 Y200F 1.7 1.5 G460D 1.2 1.1 Y200C 1.7 1.6G460E 1.3 1.2 Y200V 1.8 1.4 T463C 1.1 1.2 Y200T 1.8 1.6 T463V 1.1 1.2Y200L 1.8 1.6 T463L 1.1 1.0 Y200A 1.9 1.7 T463D 1.2 1.2 Y200G 2.0 1.8T463H 1.2 1.3 Y200S 2.2 2.0 I464V 1.1 1.0 M202T 1.8 1.5 N465G 1.1 1.1Y203F 1.5 1.3 N465L 1.4 1.3 Y203G 1.5 1.3 N465R 1.5 1.6 Y203C 1.5 1.2N465Q 1.5 1.4 Y203I 1.6 1.4 Q466L 1.1 1.1 Y203Q 1.6 1.4 Q466C 1.1 1.1Y203H 1.7 1.4 Q466E 1.1 1.2 Y203W 1.8 1.3 D467C 1.2 1.1 Y203T 1.9 1.4D467V 1.4 1.3 Y203L 2.0 1.6 W469M 1.0 1.4 Y203M 2.0 1.7 W469E 1.2 1.4Y203S 2.1 1.2 W469T 1.3 1.3 Y203V 2.1 1.7 H471Q 1.3 1.1 A204P 1.5 1.3F473Y 1.2 1.1 A204R 1.5 1.4 F473C 1.2 1.1 A204C 1.5 1.3 F473R 1.2 1.3A204H 1.6 1.4 N475T 1.2 1.1 A204W 1.6 1.4 N475H 1.4 1.3 A204F 1.7 1.4G477Q 1.6 1.4 A204L 1.8 1.6 G477F 1.6 1.5 A204Y 1.8 1.5 G477K 1.6 1.5A204D 2.0 1.6 G477L 1.8 1.8 A204G 2.1 1.7 V481Q 1.2 1.1 D205C 1.8 1.6V481S 1.6 1.5 W482F 1.1 1.0

The variants that showed an IF of at least 1.0 for at least one of theModel detergents are considered to have an improved performance whencompared to the parent alpha-amylase, i.e. the alpha-amylase having theamino acid sequence set forth in SEQ ID NO: 3.

Example 3 Stability of Variants

The variants generated as described and listed in Example 1, werefurther evaluated for stability in Model detergents.

Substrate: Phadebas Tablets (Magle Life Sciences)

-   1 tablet was suspended in 10 ml of the detergent solutions.    Assay Procedure:

20 ul of culture supernatant was transferred into two 96Well platesnamed as Unstress and Stress. To this 80 ul of detergent mixed with EDTAwas added, and mixed well. The Unstressed plate was incubated at 4° C.for 16 hrs and Stressed plates were incubated at 43° C. for 16 hrs. TheUnstress and Stress samples were diluted 20× in buffer. 20 ul of thediluted culture was added to the pre-dispensed substrate plate and mixedwell. The plate was incubated for 20 min at 25° C. with shaking (900rpm). After the incubation the plate was allowed to settle for 5 mins.50 ul of the supernatant was transferred into 384 well plates and theabsorbance was measured at 620 nm.

The stability was calculated as % Residual activity of the ratio betweenStress and Unstress sample (% RA=stress/unstress*100). The ImprovementFactor (IF) was calculated as: % RA of variant / % RA of WT.

TABLE 3 The stability results obtained are listed below SubstitutionStability H1V 1.6 H1L 1.5 H2T 1.2 D3R 1.3 D3Y 1.4 D3W 1.4 D3S 1.5 D3F1.5 T5L 1.3 T5F 1.2 T5W 1.2 G7F 1.6 G7V 1.3 G7E 1.4 G7E 1.7 G7D 1.2 G7L1.3 G7A 1.7 I9M 1.0 I9L 1.2 Q11L 1.1 Q11V 1.0 Q11M 1.0 E14Q 1.4 W15G 1.1W15S 1.0 W15I 1.2 N16R 1.5 V17D 1.5 V17G 1.5 V17E 1.5 V17Q 1.6 V17A 1.6V17T 1.7 V17L 1.7 N19G 1.1 N19S 1.1 Q22C 1.2 Q22A 1.5 Q22Y 1.5 H23W 1.2W24F 1.2 N25V 1.4 N25R 1.2 N25L 1.2 N25T 1.3 R26L 1.3 R26E 1.3 H28E 1.6H28R 1.4 H28M 1.5 H28W 1.7 H28K 1.8 N29G 1.3 N29K 1.1 N29V 1.3 N29W 1.4N30S 1.1 N30R 1.1 A31S 1.0 A31G 1.0 A31R 1.0 N33D 3.3 L34Y 1.0 K35L 1.0N36V 1.7 N36P 1.7 N36W 1.8 A37E 1.4 G38M 1.1 G38Y 1.3 G38K 1.3 I39L 1.2I39V 1.2 T40Q 1.7 T40A 1.3 T40S 2.3 A41Q 1.2 I42V 1.1 I42L 1.2 I44T 1.2I44L 1.1 I44V 1.1 A47G 1.0 A47T 1.0 W48C 1.7 W48I 2.0 W48H 1.8 W48A 1.8K49Q 1.1 S52M 1.6 S52V 1.8 S52C 1.5 S52F 1.5 S52A 1.6 S52D 1.7 S52K 1.7S52N 1.8 S52E 1.8 S52T 1.8 Q53V 1.5 Q53L 1.8 Q53M 1.6 N54T 1.7 N54L 1.8N54I 1.9 D55G 1.8 V56L 1.7 G59E 1.7 G59S 1.4 A60V 1.6 A60F 2.4 L63C 1.2L63G 1.2 L63A 1.3 E68S 1.3 E68M 1.3 E68C 1.3 E68T 1.4 E68V 1.4 E68Q 1.4E68Y 1.4 G73C 1.2 T74V 1.2 T74L 1.3 T74N 1.0 V75T 2.4 V75E 1.4 R76V 2.6T77V 1.1 T81G 1.2 K82G 1.0 A83Q 1.2 A83R 1.2 A83S 1.2 A83F 1.2 A83V 1.2E84M 1.0 E84W 1.1 E86W 1.3 E86R 1.3 I89A 1.0 I89L 1.1 R90L 1.3 S91A 1.1S91G 1.2 S91V 1.1 S91T 1.3 L92C 1.2 L92F 1.1 N95E 1.8 N95S 1.8 N95G 3.5N95D 1.7 N95R 1.8 G96R 1.1 I97V 1.1 I97L 1.3 Q98V 1.3 Q98L 1.4 Q98F 1.1Q98W 1.3 Q98P 1.3 Q98H 1.4 V99G 1.6 V99E 2.6 Y100W 1.2 V103A 1.0 V103S1.4 V103T 1.4 H107M 1.2 K108V 1.4 G109D 1.5 G109F 1.6 G110C 1.4 A111S1.8 D112V 1.1 D112F 1.2 D112C 1.3 D112K 2.9 F113M 1.2 F113V 1.4 F113E1.2 F113K 1.8 T114S 1.2 T114V 1.4 T114L 1.5 R116Q 1.5 R116K 1.6 V117G1.5 V117I 1.7 V117M 1.7 Q118V 1.5 Q118G 1.5 Q118E 1.6 Q118F 1.7 Q118R1.8 V120C 1.1 V120A 1.1 V120N 1.2 V122L 3.2 N123K 1.5 N123V 1.5 P124R1.3 P124G 1.5 N126G 1.1 N126A 1.1 N126R 1.1 N126T 1.7 N128S 1.7 N128E1.7 N128P 1.5 N128G 1.5 N128A 2.0 Q129E 1.3 V131I 1.6 G133A 1.3 G133D1.4 G133R 1.2 Q136E 1.8 Q136W 2.0 Q136G 2.0 I137V 1.5 E138A 1.4 E138Y1.3 E138T 1.5 E138L 2.2 A139S 2.2 A139G 2.0 A139V 2.0 A139T 2.3 A139P2.6 W140H 1.3 T141S 1.8 T141C 1.3 T141G 1.5 G142R 1.5 G142A 1.9 F143M1.4 F143L 1.8 F143I 1.3 N144M 1.4 N144K 1.4 N144G 1.6 N144R 1.8 F145A2.5 F145N 1.8 F145Q 1.8 F145T 2.0 F145H 2.1 P146L 1.7 P146M 1.6 P146T1.8 G149Y 1.8 G149L 1.8 G149A 1.9 Q151L 3.7 S153G 1.3 S153T 1.5 S154M2.3 S154E 2.4 S154A 2.4 S154V 2.4 F155W 1.2 K156F 2.1 K156Q 1.4 K156L2.0 K156S 2.1 K156G 2.0 K156N 1.2 K156T 1.2 R158S 1.0 Y160W 1.1 Y160R1.1 Y160H 1.5 H161C 1.1 T165I 1.5 T165V 1.1 T165E 1.1 D166S 1.6 Q169L1.6 Q169S 1.6 Q169V 1.8 Q169F 2.2 S170A 2.5 S170V 2.3 S170Q 1.8 S170W1.8 S170G 1.8 S170F 1.8 S170T 1.9 S170L 2.2 R171A 1.6 R171M 1.6 Q172L1.8 Q172E 1.8 Q172C 1.5 Q172R 1.8 Q172G 1.9 Q172W 2.1 L173I 1.2 L173A1.2 A174V 1.4 A174L 1.4 A174M 1.4 A174K 1.6 N175G 1.2 N175S 1.4 R176Y1.5 R176M 1.6 R176T 1.8 R176K 2.2 Y178W 1.1 K179L 1.5 R181A 1.8 R181G1.8 R181D 1.5 K185G 3.5 A186S 2.1 A186D 1.7 A186T 1.7 A186Q 1.8 A186V 2T193S 1.6 T193D 1.5 T193R 1.5 T193G 2.6 N195T 1.5 N195F 1.9 G196P 1.2Y198A 1.0 Y198G 1.2 Y203W 1.6 Y203L 2.0 D205R 1.3 D205A 1.6 D205L 2.9V206A 2.1 V206S 2.1 V206K 2.4 D209S 2.2 D209M 2.2 D209V 2.6 D209Q 1.5D209L 1.9 D209I 2.5 E212G 1.7 E212I 2.1 E212Y 1.4 E212R 1.4 E212C 1.5E212K 1.8 V213C 1.2 V213W 1.9 V213R 1.6 V213F 1.6 I214V 1.3 N215C 1.7N215T 2.0 L217S 1.3 L217A 1.2 L217W 1.3 N218S 1.4 N218D 1.6 N218R 1.6N218L 1.8 N218M 2.0 R219L 1.1 W220L 1.5 W220I 1.6 W220P 1.7 W220E 1.7W220R 1.8 W220A 1.8 W220S 1.8 G221S 1.0 G221A 1.5 V222E 1.1 V222L 1.1W223F 1.1 W223L 1.2 N226C 2.4 N226M 1.9 N226E 2.0 N226K 2.2 N226R 2.3T227L 1.3 T227Y 1.2 T227R 1.2 L228F 1.2 L228M 1.0 D231P 1.2 F233T 2.5F233P 2.6 F233M 3.2 V238A 1.2 H240W 1.3 K242A 1.9 F245G 1.0 F245A 1.0R247Y 1.2 R247G 1.3 R247M 1.3 D248G 1.6 D248R 1.6 W249F 1.1 L250C 1.4L250F 1.3 L250Y 1.6 G251K 1.6 G251M 1.7 G251Q 1.9 H252C 1.4 H252T 1.4H252F 1.5 H252V 1.6 V253A 1.0 V253L 1.1 T257V 1.3 G258P 1.1 G258F 1.1G258D 3.2 K259L 1.3 K259A 1.3 K259S 1.3 K259D 1.4 K259C 1.4 N260W 1.5N260S 1.4 N260R 1.5 L261T 1.3 V264L 1.0 W268Y 1.1 K269W 1.8 K269Q 1.3K269R 1.3 L272M 1.2 G273E 1.5 L275G 1.2 L275E 1.2 L275A 1.4 L275R 1.4L275K 1.4 E276L 1.6 N277D 1.5 L279Q 1.4 L279M 1.3 S280R 1.0 S280E 1.0S280D 1.1 K281A 1.4 T285W 1.4 T285M 1.5 T285P 2.1 M286L 1.2 M286C 1.2P292L 1.8 P292G 1.7 L293T 1.3 L293G 1.5 L293Y 1.3 L293Q 2.1 H294W 1.2H294Q 1.2 H294S 1.5 Y295I 1.1 Y295A 1.2 N296S 1.3 N296K 1.4 N296H 1.9L297W 1.4 L297H 1.2 Q299S 1.4 Q299L 1.8 Q299R 2.1 Q299A 1.4 Q299H 1.4Q299P 1.6 A300C 1.2 N302S 1.2 S303A 1.1 S303M 1.3 S304G 1.6 S304L 1.6S304F 1.7 S304A 1.8 S304M 1.9 G305Q 1.1 G305F 1.9 G305I 2.0 N306V 2.0N306L 1.8 N306D 1.9 N306S 2.0 N306T 2.0 R310A 1.4 R310T 1.5 N311L 1.2N311R 1.3 N311C 1.8 N311Y 1.8 L312F 2.0 L312V 1.6 L312I 1.4 L313M 1.4L313I 1.4 G315V 1.3 G315T 1.2 G315R 1.3 G315F 1.4 T316S 1.3 T316C 1.2T316G 1.2 V318L 1.0 Q319P 1.3 Q319H 1.6 Q319D 1.6 Q319F 1.7 R320V 1.6R320C 1.0 R320A 1.1 R320S 1.1 H321T 1.4 H321G 1.2 H321R 1.5 H321S 1.6V326G 1.0 V326A 1.0 V326C 1.0 V326T 1.1 T327V 2.2 T327L 1.3 T327Q 1.1T327E 1.6 F328M 1.1 F328C 1.1 V329S 1.0 V329C 1.1 V329I 1.1 T334A 1.4T334S 1.6 P336L 2.0 P336W 1.5 P336S 1.6 P336K 1.8 G337V 1.1 G337F 1.1E338T 1.0 E338A 1.6 E338H 2.6 E341P 1.4 E341M 1.7 E341F 1.9 E341K 1.9E341S 2.2 S342A 1.2 V344L 1.4 Q345L 1.0 Q345W 1.0 Q345N 1.0 G346R 1.0G346S 1.7 W347L 1.9 W347R 1.6 W347V 1.6 W347C 1.7 F348P 1.0 F348M 1.3P350S 1.1 P350Q 1.3 P350D 1.1 P350E 1.1 L351W 1.2 L351H 1.5 L351Q 1.6A354C 2.1 T355L 1.1 T355Y 1.2 T355F 1.2 L357H 1.0 T358L 1.3 E360G 1.2E360A 1.2 E360M 1.4 Q361V 1.0 Q361G 1.0 Q361D 1.0 Q361S 1.3 Y363L 1.4Y363R 1.5 Y363V 1.9 Q365M 1.0 Q365L 1.1 Q365T 1.1 Q365C 1.1 Q365S 1.3V366I 1.0 V366C 1.0 F367L 1.2 F367S 1.2 F367A 1.3 Y368H 1.3 G369L 1.3Y372A 1.1 G373T 1.4 G373Q 1.5 G373W 1.5 G373R 1.6 I374Y 1.5 I374S 1.5I374M 1.5 I374L 1.6 P375E 1.5 P375S 1.5 P375V 1.6 P375R 1.6 S376R 1.5S376L 1.1 S376V 1.3 S376I 1.4 S376Q 1.4 S376F 1.5 S376E 1.7 S376T 1.7D377S 1.0 D377G 1.3 D377T 1.9 V379L 2.0 V379S 1.5 P380V 1.1 P380Y 1.1S381V 1.7 Y382K 1.3 Y382R 2.7 R383V 1.1 Q384S 1.2 Q384V 1.2 Q384T 1.3Q384G 1.3 Q384E 1.5 I386L 1.1 I386M 1.1 I386W 1.2 I386V 1.2 D387Q 1.2D387L 1.4 P388S 1.5 P388R 1.5 P388K 1.7 P388L 1.7 L389A 1.6 L389V 1.6L389F 1.7 L389M 1.8 L390V 1.5 L390T 1.2 L390N 1.3 L390H 1.3 L390C 1.5K391Q 1.3 A392I 1.4 A392M 1.3 A392T 1.4 Q394N 1.5 Q394L 1.7 Q394T 1.9Q394M 1.9 Q395E 1.2 Q395K 1.4 Q395R 1.5 Q395V 1.0 Q395S 1.0 Q395M 1.0Y396R 1.2 Y396W 1.3 A397S 1.3 Y398S 1.3 Y398C 1.4 Y398N 1.4 Y398K 1.6R400E 1.5 R400I 1.5 H402L 1.1 H402D 1.1 H402R 1.3 H402T 1.5 D403P 1.1D403Q 1.1 D403I 1.2 Y404K 1.3 Y404F 1.7 Y404W 1.8 F405G 1.4 F405A 1.2D406V 1.1 D406T 1.1 D406M 1.1 D406S 1.2 D409G 1.2 V410L 1.3 V410E 2.1T414A 1.0 R415A 1.0 R415Q 1.3 E416V 1.4 E416L 1.2 E416A 1.3 E416Q 1.5N418M 1.5 N418G 1.6 N418R 1.7 N418F 1.9 A419M 1.5 A419G 1.2 A419V 1.4A419L 1.5 A419Q 1.6 A419R 1.8 S420M 1.8 H421T 1.1 P422Y 1.7 P422H 1.8P422D 1.8 P422R 1.9 S424L 1.2 G425V 1.2 L426S 1.2 L426A 1.3 A427S 1.3M430L 1.6 M430A 1.2 M430T 1.3 S431A 1.5 S431T 1.2 S431F 2.4 D432Q 1.2D432P 1.2 G435N 1.2 G435M 1.3 G435I 1.4 G435S 1.6 W439E 1.2 W439A 1.4Y441K 1.2 Q445R 1.6 K446G 1.4 K446T 1.1 K446P 1.3 A447R 1.1 A447S 1.5G448R 1.0 V450T 1.1 V450Q 1.1 V450R 1.2 W451L 1.0 H452C 1.1 H452G 1.1H452Y 1.1 H452R 1.1 H452V 1.4 M454Q 1.3 M454S 1.5 G456Y 1.2 G456K 1.1G456L 1.1 S459E 1.1 S459Q 1.2 S459R 1.1 T463M 1.1 T463A 1.1 T463Q 1.1N465L 1.1 N465R 1.1 D467C 1.1 F473E 1.2 F473G 1.3 F473T 1.4 F473V 1.5N475G 1.5 G476F 1.1 W482F 1.0

The variants having an IF of at least 1.0 were considered to have animproved stability compared to the parent alpha-amylase, i.e. thealpha-amylase having an amino acid sequence as set forth in SEQ ID NO:3.

Example 4 Mean Hit Values of Variants

As can be seen from FIG. 1-3, a large number of variants have beengenerated and tested for specific activity in Model A and Model Xdetergent compositions as well as for stability. In particular, FIG. 1shows the Improvement Factor (IF) as a measure for the specific activityof the variants in Model A detergent composition, FIG. 2 shows the IF asa measure of the specific activity of the variants in Model X detergentcomposition, and FIG. 3 shows the IF as a measure for the stability ofthe variants in Model A detergent composition. A mean hit value for eachposition which has been subject for modifications have been calculated(see Table 4).

TABLE 4 Mean hit value for each position Position Model A Model XStability 1 1.2 1.2 1.2 2 1.2 1.1 1.5 3 1.0 1.1 1.2 4 1.0 1.1 1.1 5 1.11.2 1.1 6 1.3 1.4 1.1 7 1.4 1.3 1.3 8 1.1 1.1 1.9 9 1.1 1.2 1.7 10 1.11.2 1.2 11 1.1 1.1 1.6 12 1.1 1.1 2.2 13 1.2 1.1 1.5 14 1.5 1.1 1.5 151.2 1.1 1.2 16 1.2 1.2 1.2 17 1.2 1.2 1.4 18 1.2 1.2 1.8 19 1.3 1.3 1.220 1.0 1.0 1.5 21 1.1 1.1 1.0 22 1.2 1.2 1.2 23 1.2 1.2 1.2 24 1.7 1.61.0 25 1.2 1.2 1.1 26 1.3 1.3 1.3 27 1.1 1.1 1.3 28 1.1 1.1 1.3 29 1.11.1 1.1 30 1.1 1.1 1.1 31 1.1 1.2 1.0 32 1.2 1.2 33 2.5 1.0 1.5 34 1.21.1 1.4 35 1.1 1.1 1.0 36 1.1 1.1 1.3 37 1.1 1.1 1.1 38 1.2 1.5 1.1 391.4 1.4 1.1 40 1.1 1.0 1.3 41 1.2 1.1 1.2 42 1.0 1.2 1.6 43 1.2 1.2 1.944 1.1 1.1 1.3 45 1.6 1.6 1.4 46 1.4 1.5 1.6 47 1.0 1.1 1.3 48 1.3 1.32.0 49 1.0 1.4 1.8 50 1.3 1.4 1.3 51 1.3 52 1.2 1.2 1.4 53 1.2 1.2 1.354 1.1 1.1 1.3 55 1.3 1.3 1.1 56 1.2 1.2 1.6 57 1.1 1.4 1.1 58 1.2 1.11.8 59 1.2 1.2 1.5 60 1.3 1.3 1.6 61 1.2 1.6 2.0 62 1.4 1.1 2.1 63 1.11.3 1.1 64 1.2 1.2 1.7 65 1.4 1.7 1.8 66 1.1 1.1 1.2 67 1.7 1.2 1.8 681.1 1.1 1.3 69 1.1 1.1 1.5 70 1.2 1.2 1.5 71 1.3 1.2 2.2 72 1.3 1.3 1.373 1.2 1.2 1.2 74 1.2 1.2 1.6 75 1.1 1.1 1.3 76 1.2 1.2 1.8 77 1.1 1.11.8 78 1.3 1.1 1.9 79 1.3 1.1 1.7 80 2.0 1.7 1.1 81 1.0 1.0 1.1 82 1.41.3 1.6 83 1.1 1.1 1.2 84 1.4 1.0 1.9 85 1.0 1.0 1.2 86 1.3 1.0 1.1 871.2 1.1 1.3 88 1.1 1.1 1.5 89 1.3 1.2 1.3 90 1.3 1.3 1.3 91 1.2 1.2 1.192 1.4 1.2 1.9 93 1.2 1.2 1.6 94 1.1 1.1 1.0 95 1.2 1.1 1.6 96 1.0 1.11.1 97 1.1 1.1 1.2 98 1.3 1.2 1.1 99 1.2 1.2 1.4 100 1.3 1.3 1.4 101 1.21.2 1.6 102 1.3 1.1 2.0 103 1.1 1.1 1.5 104 1.1 1.1 1.9 105 1.5 1.1 2.1106 1.0 2.0 107 1.2 1.1 1.7 108 1.2 1.3 1.2 109 1.4 1.4 1.3 110 1.5 1.41.1 111 1.4 1.1 1.3 112 1.6 1.4 1.7 113 1.4 1.3 1.6 114 1.2 1.2 1.2 1151.2 1.1 116 1.4 1.4 1.4 117 1.7 1.3 1.3 118 1.3 1.3 1.4 119 1.3 1.2 1.4120 1.1 1.2 1.2 121 1.3 1.3 1.0 122 1.2 1.1 1.6 123 1.2 1.2 1.4 124 1.31.2 1.2 125 1.1 1.1 1.8 126 1.2 1.2 1.5 127 1.6 1.5 1.2 128 1.3 1.2 1.3129 1.2 1.2 1.5 130 1.2 1.2 1.8 131 1.2 1.2 1.3 132 1.3 1.3 1.4 133 1.21.2 1.4 134 1.2 1.2 1.6 135 1.5 1.5 1.5 136 1.2 1.2 1.6 137 1.3 1.2 1.2138 1.0 1.0 1.3 139 1.3 1.2 1.7 140 1.4 1.3 1.0 141 1.3 1.3 1.9 142 1.21.2 1.3 143 1.2 1.2 1.7 144 1.2 1.1 1.2 145 1.2 1.2 2.3 146 1.2 1.2 1.3147 1.2 1.2 1.1 148 1.3 1.1 1.4 149 1.2 1.2 1.2 150 1.2 1.1 2.2 151 1.31.3 1.7 152 1.3 1.1 1.1 153 1.1 1.1 2.0 154 1.1 1.1 2.5 155 1.2 1.2 1.2156 1.4 1.4 1.8 157 1.1 1.1 1.2 158 1.5 1.5 1.1 159 1.3 1.3 160 1.4 1.41.2 161 1.0 1.0 1.8 162 1.2 1.2 1.4 163 1.4 1.4 2.0 164 1.3 1.3 165 1.41.3 1.1 166 1.5 1.3 1.5 167 1.3 1.4 1.6 168 1.5 1.2 1.5 169 1.2 1.2 1.8170 1.3 1.2 1.6 171 1.8 1.7 1.6 172 1.1 1.1 1.6 173 1.3 1.3 1.4 174 1.21.2 1.1 175 1.2 1.2 1.6 176 1.6 1.5 1.6 177 1.4 1.2 2.2 178 1.2 1.2 1.8179 1.6 1.5 1.3 180 1.1 1.1 1.5 181 1.5 1.6 1.3 184 1.1 1.1 1.0 185 1.71.5 1.0 186 1.2 1.1 1.3 187 1.3 1.2 1.8 188 1.3 1.2 1.8 189 1.1 1.1 1.3190 1.2 1.1 2.4 191 1.2 1.1 1.3 192 1.1 1.0 2.3 193 1.1 1.1 1.3 194 1.11.1 1.4 195 1.0 1.1 1.4 196 1.1 1.1 1.1 197 1.1 1.1 1.1 198 1.0 1.6 1.1199 1.3 1.3 1.5 200 1.0 1.0 2.1 201 1.0 1.2 202 1.2 1.1 1.0 203 1.5 1.31.3 204 1.0 1.1 1.5 205 1.9 1.4 2.1 206 1.1 1.1 2.3 207 1.1 1.1 2.0 2081.0 1.0 209 1.1 1.2 1.5 210 1.3 1.4 1.3 211 1.2 1.3 1.9 212 1.0 1.1 1.7213 1.1 1.1 1.5 213 1.1 1.2 1.8 214 1.1 1.0 1.3 215 1.3 1.2 1.7 216 1.11.1 1.3 217 1.2 1.2 1.3 218 1.1 1.1 1.3 219 1.4 1.3 1.3 220 1.3 1.4 2.0221 1.1 1.1 1.9 222 1.1 1.1 1.1 223 1.5 1.4 1.2 224 1.1 1.1 1.6 225 1.11.1 1.3 226 1.1 1.1 1.7 227 1.1 1.1 1.4 228 1.1 1.1 1.9 229 1.1 1.1 1.2230 1.1 1.1 2.0 231 1.1 1.0 2.0 232 1.0 1.0 1.7 233 1.1 1.2 1.5 234 1.31.0 2.1 235 1.1 1.1 1.4 236 2.6 1.9 237 1.1 1.1 2.0 238 1.3 1.2 1.2 2391.1 1.1 1.6 240 2.0 241 1.1 1.0 1.8 242 1.3 1.4 2.0 243 1.1 1.1 2.0 2441.0 1.0 1.4 245 1.5 1.5 1.8 246 1.1 1.1 1.4 247 1.2 1.2 1.2 248 1.1 1.11.4 249 1.1 1.1 1.6 250 1.1 1.1 1.3 251 1.1 1.1 1.2 252 1.1 1.0 1.2 2531.2 1.2 1.8 254 1.1 1.1 1.0 255 1.0 1.0 1.4 256 1.1 1.1 1.8 257 1.0 1.02.2 258 1.1 1.1 1.0 259 1.1 1.1 1.1 260 1.1 1.1 1.2 261 1.1 1.1 1.1 2621.3 1.5 1.1 263 1.2 1.2 1.1 264 1.1 1.1 1.7 265 1.2 1.2 1.4 266 1.3 1.21.7 267 1.1 1.1 2.1 268 1.1 1.0 1.8 269 1.2 1.3 1.4 269 1.3 1.2 1.5 2701.2 1.3 1.2 271 1.1 1.3 1.6 272 1.3 1.1 1.6 273 1.1 1.2 1.2 274 1.2 1.21.1 275 1.1 1.1 1.7 276 1.1 1.2 1.4 277 1.0 1.0 2.6 278 1.1 1.0 1.9 2791.2 1.2 1.4 280 1.1 1.1 1.4 281 1.1 1.1 1.4 282 1.0 1.1 1.8 283 1.1 1.11.4 284 1.2 1.2 1.1 285 1.0 1.0 1.2 286 1.1 1.1 1.2 287 1.2 1.3 1.6 2881.1 1.2 1.4 289 1.3 1.5 1.2 290 1.2 2.1 2.2 291 1.1 1.1 1.4 292 1.1 1.11.6 293 1.2 1.2 1.2 294 1.1 1.1 1.4 295 1.1 1.1 1.1 296 1.2 1.2 1.4 2971.1 1.1 1.7 298 1.0 1.2 1.4 299 1.2 1.1 1.7 300 1.1 1.1 1.5 301 1.1 1.21.2 302 1.2 1.1 1.2 303 1.1 1.1 1.2 304 1.0 1.0 1.5 305 1.1 1.2 1.1 3061.0 1.1 1.4 307 1.0 1.0 1.5 308 1.5 1.6 1.9 309 1.0 1.1 1.7 310 1.2 1.11.4 311 1.2 1.2 1.3 312 1.2 1.3 1.5 313 1.1 1.0 1.5 314 1.2 1.1 1.2 3151.1 1.1 1.2 316 1.0 1.1 1.4 317 1.1 1.3 2.1 318 1.1 1.1 1.4 319 1.3 1.22.6 320 1.3 1.1 1.5 321 1.2 1.2 1.3 322 1.2 1.4 1.3 323 1.1 1.2 1.2 3241.2 1.2 1.5 325 1.1 1.1 2.1 326 1.0 1.1 1.3 327 1.1 1.1 1.3 328 1.1 1.71.5 329 1.1 1.0 1.5 330 2.5 331 1.2 1.1 1.1 332 1.2 1.4 1.3 333 2.4 3341.1 1.1 1.6 335 1.1 1.1 1.3 336 1.2 1.2 1.3 337 1.5 1.2 1.3 338 1.0 1.11.3 339 1.1 1.4 2.0 340 1.0 1.0 1.6 341 1.2 1.1 1.4 342 1.1 1.1 1.3 3431.2 1.1 1.6 344 1.1 1.1 1.0 345 1.1 1.1 1.0 346 1.1 1.2 1.3 347 1.2 1.31.5 348 1.2 1.2 1.1 349 1.7 1.2 1.7 350 1.3 1.3 1.7 351 1.2 1.2 1.5 3521.2 1.4 1.8 353 1.0 1.0 2.0 354 1.1 1.1 1.2 355 1.0 1.0 1.6 356 1.1 1.11.2 357 1.1 1.1 1.0 358 1.1 1.2 1.6 359 1.1 1.0 1.4 360 1.1 1.1 1.1 3611.1 1.2 1.3 362 1.3 1.3 1.4 363 1.2 1.2 1.3 364 1.2 1.2 1.0 365 1.0 1.11.5 366 1.1 1.1 1.1 367 1.5 1.2 1.8 368 1.2 1.2 1.1 369 1.7 1.4 1.6 3701.2 1.2 2.1 371 1.1 1.1 1.1 372 1.1 1.1 1.2 373 1.1 1.1 1.1 374 1.1 1.11.2 375 1.0 1.1 1.2 376 1.2 1.2 1.1 377 1.2 1.2 1.4 378 1.1 1.1 1.9 3791.1 1.1 1.5 380 1.2 1.2 1.2 381 1.2 1.1 2.2 382 1.2 1.2 1.8 383 1.1 1.11.7 384 1.0 1.1 1.1 385 1.1 1.1 1.2 386 1.2 1.1 1.3 387 1.1 1.1 1.2 3881.1 1.1 1.2 389 1.2 1.2 1.4 390 1.1 1.1 1.3 391 1.1 1.1 1.3 392 1.0 1.11.1 393 1.2 1.1 1.2 394 1.1 1.1 1.0 395 1.2 1.1 1.5 396 1.1 1.1 1.2 3971.1 1.1 1.1 398 1.0 1.0 1.2 399 1.1 1.2 1.3 400 1.2 1.1 1.2 401 1.2 1.11.1 402 1.2 1.2 1.2 403 1.0 1.1 1.4 404 1.0 1.0 1.1 405 1.1 1.2 1.4 4061.3 1.3 1.3 407 1.2 1.3 1.7 408 1.2 1.1 1.9 409 1.5 1.4 2.1 410 1.2 1.21.3 411 1.1 1.1 1.5 412 1.3 1.2 2.0 413 1.4 1.2 1.6 414 1.1 1.1 1.9 4151.1 1.1 1.7 416 1.3 1.2 1.2 417 1.0 1.2 1.4 418 1.2 1.2 1.3 419 1.1 1.11.3 420 1.0 1.0 1.7 421 1.1 1.1 1.3 422 1.0 1.5 1.5 423 1.0 1.1 1.2 4241.1 1.0 1.4 425 1.2 1.2 1.9 426 1.0 1.0 1.1 427 1.1 1.3 1.1 428 1.1 1.22.0 429 1.1 1.2 2.9 430 1.0 1.0 1.3 431 1.1 1.2 1.3 432 1.0 1.9 1.5 4331.1 1.1 1.0 434 1.2 1.2 1.2 435 1.3 1.3 1.1 436 1.1 1.2 1.1 437 1.1 1.11.1 438 1.0 1.0 1.1 439 1.1 1.2 1.4 440 1.1 1.2 1.6 441 1.0 1.0 1.0 4421.1 1.1 1.2 443 1.1 1.1 444 1.1 1.1 1.6 445 1.1 1.0 1.2 446 1.0 1.0 1.1447 1.1 1.1 1.1 448 1.1 1.1 1.0 449 1.1 1.1 1.2 450 1.0 1.0 1.2 451 1.11.1 1.7 452 1.0 1.1 1.1 453 1.1 1.3 2.3 454 1.1 1.0 1.3 455 1.1 1.1 1.6456 1.1 1.1 1.1 457 1.0 1.1 1.2 458 1.1 1.1 1.1 459 1.1 1.1 1.2 460 1.11.1 1.3 461 1.1 1.1 1.1 462 1.1 1.1 1.1 463 1.0 1.0 1.1 464 1.1 1.1 4651.1 1.1 1.4 466 1.0 1.0 2.2 467 1.1 1.1 1.7 468 1.3 1.1 1.9 469 1.1 1.1470 1.0 1.0 1.5 471 1.1 1.1 1.2 472 2.2 1.2 1.9 473 1.2 1.1 1.2 474 1.21.3 1.8 475 1.1 1.1 1.9 476 1.4 1.3 1.3 477 1.4 1.3 1.2 478 1.0 1.1 1.2479 1.0 1.0 2.2 480 1.1 1.2 1.7 481 1.0 1.1 1.0 482 1.1 1.2 1.9 483 1.11.1 1.0

As can be seen from the table above, each of the tested positions havean IF of at least 1.0 for at least one of the conditions the variantshave been tested for, i.e. specific activity in Model A and/or Model Xdetergent compositions, and/or stability in Model A detergentcomposition.

Example 5 Specific Activity and Stability of Variants of the Invention

In order to determine whether the variants generated as described andlisted in Example 2 have a maintained or even improved activity, thevariants were evaluated by the Phadebas assay. The following detergentcompositions were prepared;

Preparation of Model X (0.175%):

1:2 molar ratio of CaCl2 and MgCl2 stock solution with 6000 dH (waterhardness).

104.9 g of CaCl₂.2H₂O (0.713M) was weighed into 1 liter bottle and tothis 500 ml of type I water was added and stirred well. To this 72.5 gof MgCl2.6H2O (0.357M) was weighed and added, dissolved well and thefinal volume was made upto 1000 ml with type I water.

0.535 M Solution of NaHCO₃

44.9 g of Sodium Hydrogen carbonate was dissolved in 100 ml of type Iwater.

Model X Detergent with X Ionics with a Water Hardness of 12 (12° dH)

1.75 g of Model X detergent (as described above) was weighed andtransferred into 1 litre bottle and to this 800 ml of type I water wasadded and mixed well. To this 35 mg of X-ionics was added and mixedwell. To adjust the water hardness to 12° dH, 2 ml of 1:2 molar ratio ofCaCl₂ and MgCl₂ stock solution with 6000° dH, 6 ml of 0.535 Molarsolution of NaHCO₃ was added and mixed well. Finally the volume was madeup to 1000 ml and the mixture was stirred for 10 min.

Preparation of Model A (0.33%):

4:1 Molar Ratio of CaCl2 and MgCl2 Stock Solution with 6000 dH (WaterHardness)

125.8 g of CaCl2.2H2O was weighed into 1 liter bottle and to this 500 mlof type I water was added and stirred well. To this 43.8 g of MgCl2.6H2Owas weighed and added and dissolved well and the final volume was madeup to 1000 ml with type I water.

0.535 M Solution of NaHCO₃

44.9 g of Sodium Hydrogen carbonate was dissolved in 100 ml of type Iwater.

Model A Detergent with a Water Hardness of 15 (15° dH)

3.335 g of Model A detergent was weighed and transferred into 1 litrebottle and to this 865 ml of type I water was added and mixed well. Tothis 7.5 ml of 0.535M NaHCO₃ was added, mixed well and made up thevolume to 1 liter with type 1 water. To adjust the water hardness to 15°dH 2.5 ml of 4:1 molar ratio of CaCl₂.2H₂O and MgCl₂.6H₂O stock solutionwith 6000° dH was added and the mixture was stirred for 15 min.

Substrate: Phadebas Tablets (Magle Life Sciences)

1 tablet was suspended in 10 ml of the detergent solutions.

Buffer: 100 mM MOPS Buffer pH 8

Experimental Procedure

Preparation of the Mother Plates:

Colonies were picked from the transformed plate by colony picker(KBiosystems) and inoculated in 96-well culture plate comprising TBGlymedia for growth. The cultures were grown for 3 days at 37° C. and thesupernatant was recovered from the plates by centrifugation.

Preparation of the Substrate Plates:

The substrate solution was prepared by dissolving 1 tablet of phadebasin 10 ml of Model X/Model A detergent and 180 ul of the same wasdispensed into 96 Well micro titer plate using multidrop instrument withconstant stirring.

The culture supernatant was diluted to 100× with buffer and 20 ul of thediluted culture was added to the 180 ul of pre-dispensed substrate plateand mixed well. The plate was incubated for 20 min at 25 C with shaking(900 rpm). After the incubation the plate was allowed to settle for 5mins. 50 ul of the supernatant was transferred into 384 well plates andthe absorbance was measured at 620 nm. The concentration of theexpressed enzyme was determined by ELISA using specific antibodies. Thespecific activity was calculated by taking the ratio of the activity byconcentration and the hits were identified as anything higher than thespecific activity of the parent alpha-amylase. The Improvement Factor(IF) was calculated as:[Specific Activity of variant]/[Specific Activity of parentalpha-amylase].Stability of the Variants of the InventionSubstrate: Phadebas Tablets (Magle Life Sciences)

1 tablet was suspended in 10 ml of the detergent solutions.

Assay Procedure:

20 ul of culture supernatant was transferred into two 96Well platesnamed as Unstress and Stress. To this 80 ul of detergent mixed with EDTAwas added, and mixed well. The Unstressed plate was incubated at 4° C.for 16 hrs and Stressed plates were incubated at 43° C. for 16 hrs. TheUnstress and Stress samples were diluted 20× in buffer. 20 ul of thediluted culture was added to the pre-dispensed substrate plate and mixedwell. The plate was incubated for 20 min at 25° C. with shaking (900rpm). After the incubation the plate was allowed to settle for 5 mins.50 ul of the supernatant was transferred into 384 well plates and theabsorbance was measured at 620 nm.

The stability was calculated as % Residual activity of the ratio betweenStress and Unstress sample (% RA=stress/unstress*100). The ImprovementFactor (IF) was calculated as: % RA of variant/% RA of WT.

The specific activity of the variants according to the inventionobtained by the method described above are the following (amino acidsubstitutions refer to SEQ ID NO: 3) shown in Table 5 below.

TABLE 5 Specific variants of the present invention. StabilitySubstitution Model A Model X Model A G7S 0.9 0.9 1.1 G7N 0.8 0.8 1.1E14F 1.1 0.7 1.0 W15H 0.8 0.5 1.1 N29A 1.1 1.1 1.0 N36T 0.9 0.9 1.1 W48Y1.6 1.3 1.2 W48F 1.3 1.2 1.4 T51A 1.1 1.1 1.0 A60T 1.4 1.4 0.8 F113Y 1.21.3 1.0 R116W 0.7 0.7 1.0 A119W 1.1 1.0 V122I 1.4 1.2 0.0 V122F 1.1 1.00.0 S132V 1.1 1.0 0.0 S132Q 1.1 1.0 0.0 S132N 1.2 1.2 0.3 Y135F 1.3 1.50.8 Y135F 1.3 1.2 0.9 Y135E 1.5 1.7 0.8 Q151I 0.8 0.8 1.0 W159S 1.2 1.20.6 W159H 1.2 1.3 0.8 G184D 1.2 1.4 1.0 A186H 0.8 1.0 1.1 H210Q 0.9 0.91.1 H210N 1.2 1.2 1.2 K242Q 2.7 2.9 0.7 K242H 1.0 1.1 0.0 S244Q 0.7 0.71.2 N260G 1.1 0.9 1.0 A288V 0.8 0.8 1.2 Y295N 1.2 1.0 0.9 Y295F 1.3 1.21.0 S323N 1.1 1.0 1.0 E341T 1.0 1.0 1.1 W439T 0.8 0.9 1.1 W439R 1.4 1.41.0 W439N 1.4 1.4 1.0 G476Y 1.1 1.3 1.1 QG476K 1.2 1.1 0.9

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. An alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein said variant is a mature form of an alpha-amylase having amylase activity, and comprising: a) at least 85% sequence identity with the amino acid sequence set forth in SEQ ID NO: 3; b) a deletion and/or substitution at two or more positions corresponding to positions R181, G182, D183, and G184 of the amino acid sequence set forth in SEQ ID NO: 3; c) a substitution at a position corresponding to position H1 of the amino acid set forth in SEQ ID NO: 3; and d) a substitution at one or more positions selected from the group consisting of: H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E416, N418, A419, S420, H421, P422, S424, G425, L426, A427, T428, I429, M430, S431, D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides an alpha-amylase variant having an improvement factor of >1.0 for a measure of stability, and/or an improvement factor of ≥1.0 for a measure of specific activity.
 2. The alpha-amylase variant according to claim 1, wherein said variant has at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO:
 3. 3. The variant according to claim 1, comprising a substitution at one or more positions selected from the group consisting of: H2, D3, G4, T5, G7, T8, I9, Q11, Y12, F13, E14, W15, N16, P18, N19, D20, Q22, W24, N25, R26, L27, H28, N29, N30, A31, Q32, L34, K35, N36, A37, I39, T40, A41, W43, I44, P45, P46, A47, W48, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, K82, A83, E84, L85, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, D112, F113, E115, R116, V117, A119, V120, E121, V122, N123, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, I137, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, F155, K156, W157, R158, W159, Y160, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, R219, V222, W223, A225, N226, T227, L228, N229, L230, D231, F233, V238, I241, K242, F245, M246, W249, L250, H252, R254, G255, T257, G258, K259, N260, L261, F262, A263, Y267, W268, K269, N270, D271, G273, A274, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, N296, L297, Q299, A300, S301, S303, S304, G305, N306, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, A325, P336, G337, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, L357, T358, Q361, G362, Y363, Q365, V366, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, R400, H402, F405, D406, H407, W408, V410, T414, R415, E416, N418, A419, H421, G425, A427, T428, I429, M430, S431, G435, G436, S437, W439, M440, V442, R444, Q445, A447, G448, H452, M454, T455, G456, N457, S459, G460, T463, N465, D467, W469, H471, F473, N475, G477, and V481; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3, which one or more substitutions provides an improvement factor of ≥1.2 for a measure of specific activity in a detergent composition prepared according to Model A, and optionally, an improvement factor of ≥1.0 for a measure of stability.
 4. The variant according to claim 1, comprising a substitution at one or more positions selected from the group consisting of: H2, G4, T5, G7, Q11, F13, E14, W15, N16, P18, N19, D20, Q22, N25, R26, N29, N30, A31, Q32, L34, A41, P46, W48, G50, T51, S52, Q53, D55, V56, G59, A60, Y61, Y64, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, K82, A83, R87, I89, R90, S91, L92, K93, N95, Q98, V99, Y100, H107, K108, G109, D112, F113, E115, R116, V117, A119, V120, E121, V122, N123, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, I137, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, F155, K156, W157, R158, W159, Y160, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, V191, T193, E194, N197, D199, Y200, M202, Y203, A204, D205, V206, D209, H210, P211, E212, N215, L217, R219, W223, N226, V238, I241, K242, F245, G258, K259, N260, F262, A263, K269, N270, D271, G273, A274, Y278, L279, K281, W284, T285, S287, P292, N296, L297, Q299, S301, N306, L312, G315, V318, Q319, R320, H321, P322, A325, P336, G337, E341, S342, V344, G346, F348, K349, P350, L351, Q361, G362, Y363, Y368, G369, D370, Y372, G373, S376, D377, V379, P380, S381, Y382, Q384, D387, Q394, Q395, A397, R400, H402, H407, W408, V410, R415, N418, H421, S431, G435, G436, W439, M440, Q445, A447, M454, G456, N465, D467, N475, G477, and V481; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3, which one or more substitutions provides an improvement factor of ≥1.4 for a measure of specific activity in a detergent composition prepared according to Model A, and optionally, an improvement factor of ≥1.0 for a measure of stability.
 5. The variant according to claim 1, comprising a substitution at one or more positions selected from the group consisting of: G7, F13, E14, W15, N16, N19, D20, Q22, R26, N29, N30, A31, Q32, P46, W48, G50, T51, S52, Q53, D55, G59, A60, Y64, N70, Q71, K72, G73, T74, V75, K78, R90, Y100, H107, K108, G109, D112, F113, R116, V117, A119, E121, N123, N126, R127, N128, E130, V131, S132, G133, T134, Y135, I137, A139, W140, T141, G142, N144, F145, P146, G147, G149, N150, Q151, F155, K156, R158, W159, Y160, D163, G164, T165, D166, W167, S170, R171, Q172, L173, R176, Y178, K179, R181, K185, A186, W187, D188, W189, V191, E194, D199, Y200, M202, Y203, A204, D205, H210, N215, L217, R219, W223, V238, K242, F245, G258, F262, K269, N270, A274, T285, S287, N296, L312, V318, H321, P322, A325, G337, E341, S342, K349, P350, Q361, G362, Y368, G369, D377, S381, Q384, D387, R400, W408, R415, N418, G435, Q445, M454, G477, and V481; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3, which one or more substitutions provides an improvement factor of ≥1.6 for a measure of specific activity in a detergent composition prepared according to Model A, and optionally, an improvement factor of ≥1.0 for a measure of stability.
 6. The variant according to claim 1, comprising a substitution at one or more positions selected from the group consisting of: H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, P18, N19, Q22, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, G50, T51, S52, Q53, N54, D55, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, D112, F113, E115, R116, V117, A119, V120, E121, V122, N123, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, I241, K242, F245, M246, W249, L250, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, G273, A274, E276, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, A325, V326, T327, V329, N331, P336, G337, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, R400, H402, D403, F405, D406, H407, W408, V410, I411, T414, R415, E416, N418, A419, S420, H421, S424, G425, A427, T428, I429, M430, S431, G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448, V450, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; wherein the positions correspond to amino acid positions in the amino acid sequence as set forth in SEQ ID NO: 3, and wherein the one or more substitutions provides a variant having an improvement factor of ≥1.2 for a measure of specific activity in a detergent composition prepared according to Model X and optionally an improvement factor of ≥1.0 for a measure of stability.
 7. The variant according to claim 1, comprising a substitution at one or more positions selected from the group consisting of: H2, D3, T5, G7, I9, E14, W15, N16, V17, Q22, H23, W24, N25, R26, H28, N29, N33, N36, A37, G38, I39, T40, A41, I42, I44, W48, S52, Q53, N54, D55, V56, G59, A60, L63, E68, G73, T74, V75, R76, T81, A83, E86, R90, S91, L92, N95, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, R116, V117, Q118, V120, V122, N123, P124, N126, N128, Q129, V131, G133, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G149, Q151, S153, S154, F155, K156, Y160, T165, D166, Q169, S170, R171, Q172, L173, A174, N175, R176, K179, R181, K185, A186, T193, N195, G196, Y198, Y203, D205, V206, D209, H210, E212, V213, I214, N215, L217, N218, W220, G221, W223, N226, T227, L228, D231, F233, V238, H240, K242, S244, R247, D248, L250, G251, H252, T257, G258, K259, N260, L261, K269, L272, G273, L275, E276, N277, L279, K281, T285, M286, A288, P292, L293, H294, Y295, N296, L297, Q299, A300, N302, S303, S304, G305, N306, R310, N311, L312, L313, G315, T316, Q319, R320, H321, T327, T334, P336, E338, E341, S342, V344, G346, W347, F348, P350, L351, A354, T355, T358, E360, Q361, Y363, Q365, F367, Y368, G369, G373, I374, P375, S376, D377, V379, S381, Y382, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, D409, V410, R415, E416, N418, A419, S420, P422, S424, G425, L426, A427, M430, S431, D432, G435, W439, Y441, Q445, K446, A447, V450, H452, M454, G456, S459, F473, and N475; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3, which one or more substitution provides an improvement factor of ≥1.2 for a measure of stability and optionally, an improvement factor of ≥1.0 for a measure of specific activity in a detergent composition prepared according to Model A and/or Model X.
 8. The variant according to claim 1, comprising a substitution at one or more positions selected from the group consisting of: D3, G7, N16, V17, Q22, H28, N33, N36, T40, W48, S52, Q53, N54, D55, V56, G59, A60, V75, R76, N95, V99, G109, A111, D112, F113, T114, R116, V117, Q118, V122, N123, P124, N126, N128, V131, Q136, I137, E138, A139, T141, G142, F143, N144, F145, P146, G149, Q151, S153, S154, K156, Y160, T165, D166, Q169, S170, R171, Q172, A174, R176, R181, K185, A186, T193, N195, Y203, D205, V206, D209, E212, V213, N215, N218, W220, G221, N226, F233, K242, D248, L250, G251, H252, G258, N260, K269, G273, E276, N277, T285, P292, L293, H294, N296, Q299, S304, G305, N306, R310, N311, L312, Q319, R320, H321, T327, T334, P336, E338, E341, G346, W347, L351, A354, Y363, G373, I374, P375, S376, D377, V379, S381, Y382, Q384, P388, L389, L390, Q394, Q395, Y398, R400, H402, Y404, V410, E416, N418, A419, S420, P422, M430, S431, G435, Q445, A447, M454, F473, and N475; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3, which one or more substitutions provides an improvement factor of ≥1.5 for a measure of stability and optionally, an improvement factor of ≥1.0 for a measure of specific activity in a detergent composition prepared according to Model A and/or Model X.
 9. The variant according to claim 1, comprising a substitution in one or more positions selected from the group consisting of: H2, T5, G7, I9, Q11, E14, W15, N19, Q22, W24, N25, R26, H28, N29, N30, A31, L34, K35, I39, T40, A41, I42, I44, A47, W48, T51, S52, Q53, V56, G59, T74, V75, I89, R90, S91, L92, N95, I97, Q98, Y100, K108, F113, R116, V117, N126, N128, Q129, G133, A139, W140, T141, F143, F145, P146, Q151, K156, R158, H161, T165, D166, Q169, S170, R171, Q172, L173, A174, N175, K179, R181, G184, K185, A186, T193, Y203, D209, H210, E212, V213, N215, L217, N218, R219, V222, N226, T227, L228, K242, K245, L250, H252, V253, T257, G258, K259, N260, V264, K269, L279, K281, M286, P292, L293, Y295, L297, Q299, N306, L312, G315, T316, V318, R320, H321, S323, T327, V329, P336, G337, E341, S342, V344, Q345, G346, W347, P350, L351, Q361, Y363, Y368, I374, S376, V379, P380, S381, D387, L390, A392, Q395, H402, F405, T414, E416, N418, A419, M430, S431, G435, W439, K446, V450, G456, S459, N465, G476, and W482; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3, which one or more substitutions provides an improvement factor of ≥1.0 for a measure of stability and an improvement factor of ≥1.0 for a measure of specific activity in a detergent composition prepared according to Model A.
 10. The variant according to claim 1, comprising a substitution in one or more positions selected from the group consisting of: G7, Q22, N25, R26, H28, N29, W48, S52, Q53, V56, G59, T74, V75, R90, N95, Q98, K108, F113, R116, V117, N128, Q129, G133, A139, W140, T141, F143, F145, P146, Q151, K156, T165, Q169, S170, R171, Q172, A174, K179, R181, K185, A186, T193, Y203, D209, E212, N215, L217, N226, K242, K259, N260, K269, K281, P292, L293, L297, N306, L312, G315, R320, H321, P336, V344, P350, Y363, Y368, S381, D387, A392, Q395, E416, N418, and S431; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3, which one or more substitutions provides an improvement factor of ≥1.3 for a measure of stability and an improvement factor of ≥1.3 for a measure of specific activity.
 11. The variant according to claim 1, wherein said variant is a mature form of alpha-amylase having amylase activity and comprising a substitution at one or more positions selected from the group consisting of: E14, D20, Q22, G59, R116, A119, S132, G133, Y135, K156, R158, W159, Y160,K185, W187, Y202, D236, V238, K242, G337, E341, Y368, G369, D377, Y382, and K445, wherein the positions correspond to amino acids in the amino acid sequence set forth in SEQ ID NO: 3; and wherein the substitution provides at least one improved property selected from the group consisting of improved wash performance, improved detergent stability, and improved thermostability.
 12. The variant according to claim 1, wherein said variant is a mature form of alpha-amylase having amylase activity and comprising a substitution at one or more positions selected from the group consisting of: E14, D20, Q22, G59, R116, A119, S132, G133, Y135, K156, R158, W159, Y160, K185, W187, Y202, D236, V238, K242, G337, E341, Y368, G369, D377, Y382, and K445, wherein the positions correspond to amino acids in the amino acid sequence set forth in SEQ ID NO: 3; and wherein the substitution provides an alpha-amylase variant having an improvement factor of ≥1.0 for a measure of specific activity at pH 8.0 and 25° C., and an improvement factor of ≥1.0 for a measure of stability in a detergent composition and for thermostability.
 13. The variant according to claim 1, wherein the amino acid substituted in the particular position is an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, with the proviso that the amino acid substituted is different from the naturally-occurring amino acid in said particular position.
 14. The variant according to claim 1, further comprising a pairwise deletion of the amino acid residues corresponding to 181, 182, 183 and 184 of the amino acid sequence set forth in SEQ ID NO: 3, with the proviso that when the amino acids in positions corresponding to 181 and/or 184 has been substituted, the pairwise deletion is in the positions corresponding to 182 and 183 of the amino acid sequence set forth in SEQ ID NO:
 3. 15. An alpha-amylase variant, wherein said variant is a mature form having amylase activity and comprising a) a deletion and/or substitution at two or more positions corresponding to positions R181, G182, D183, and G184 of the amino acid sequence set forth in SEQ ID NO: 3; b) a substitution at a position corresponding to position H1 in the amino acid sequence set forth in SEQ ID NO: 3; and c) substitution at one or more positions selected from the group consisting of: H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E416, N418, A419, S420, H421, P422, S424, G425, L426, A427, T428, I429, M430, S431, D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO:
 3. 16. The variant according to claim 1, wherein said variant comprises a substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions.
 17. A polynucleotide encoding the variant according to claim
 1. 18. A nucleic acid construct comprising the polynucleotide according to claim
 17. 19. An expression vector comprising the polynucleotide according to claim
 17. 20. An isolated host cell comprising the polynucleotide according to claim
 17. 21. A composition comprising the variant according to claim
 1. 22. The composition according to claim 21, further comprising at least one further active component.
 23. The composition according to claim 22, wherein said further active component is an enzyme selected from the group consisting of a protease, a lipase, a cellulase, a pectate lyase, and a mannanase.
 24. The composition according to claim 21, which is a detergent composition.
 25. The composition according to claim 21, which is a liquid laundry dish wash composition, or liquid dish wash composition, an Automatic Dish Wash (ADW) liquid detergent composition, a powder laundry composition, a soap bar, a powder dish wash composition, or an ADW unit dose detergent composition.
 26. A method of improving the wash performance of a parent alpha-amylase having the amino acid sequence of SEQ ID NO: 3 or 4, or one having at least 85% sequence identity hereto, said method comprising the steps of: a) introducing a pairwise deletion and/or substitution at two or more positions corresponding to positions R181, G182, D183, and G184 of the amino acid sequence set forth in SEQ ID NO: 3; b) introducing a substitution at a position corresponding to position H1 of the amino acid sequence set forth in SEQ ID NO: 3; and c) introducing a substitution at one or more positions selected from the group consisting of: H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E416, N418, A419, S420, H421, P422, S424, G425, L426, A427, T428, I429, M430, S431, D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; said method thereby providing an alpha-amylase variant of said parent alpha-amylase, wherein said variant has at least 85%, but less than 100%, sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 3 or 4, and wherein said variant has alpha-amylase activity and improved wash performance as compared to said parent alpha-amylase.
 27. A method of improving the stability of a parent alpha-amylase having the amino acid sequence of SEQ ID NO: 3 or 4, or one having at least 85% sequence identity hereto, said method comprising the steps of: a) introducing a pairwise deletion and/or substitution at two or more positions corresponding to positions R181, G182, D183, and G184 of the amino acid sequence set forth in SEQ ID NO: 3; b) introducing a substitution at a position corresponding to position H1 of the amino acid sequence set forth in SEQ ID NO: 3; and c) introducing a substitution at one or more positions selected from the group consisting of: H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E416, N418, A419, S420, H421, P422, S424, G425, L426, A427, T428, I429, M430, S431, D432, G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; said method thereby providing an alpha-amylase variant of said parent alpha-amylase, wherein said variant has at least 85%, but less than 100%, sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 3 or 4, and wherein said variant has alpha-amylase activity and improved stability as compared to said parent alpha-amylase.
 28. The method according to claim 26, wherein said variant has at least 50% of the activity of the parent alpha-amylase having the amino acid sequence of SEQ ID NOs: 3 or
 4. 